Antibodies with modified isoelectric points

ABSTRACT

The invention relates generally to compositions and methods for altering the isoelectric point of an antibody, and in some cases, resulting in improved plasma pharmacokinetics, e.g. increased serum half-life in vivo.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/194,904, filed Jul. 29, 2011, now U.S. Pat. No. 8,637,641 whichclaims the benefit under 35 U.S.C. 119 to U.S. Provisional ApplicationSer. Nos. 61/368,962, filed Jul. 29, 2010; 61/368,969, filed Jul. 29,2010; 61/391,509, filed Oct. 8, 2010; 61/391,515, filed Oct. 8, 2010;and 61/439,263, filed Feb. 3, 2011 and are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The invention relates generally to compositions and methods for alteringthe isoelectric point of an antibody, and in some cases, resulting inimproved plasma pharmacokinetics, e.g. increased serum half-life invivo.

BACKGROUND OF THE INVENTION

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. Each chain is made up ofindividual immunoglobulin (Ig) domains, and thus the generic termimmunoglobulin is used for such proteins. Each chain is made up of twodistinct regions, referred to as the variable and constant regions. Thelight and heavy chain variable regions show significant sequencediversity between antibodies, and are responsible for binding the targetantigen. The constant regions show less sequence diversity, and areresponsible for binding a number of natural proteins to elicit importantbiochemical events. In humans there are five different classes ofantibodies including IgA (which includes subclasses IgA1 and IgA2), IgD,IgE, IgG (which includes subclasses IgG1, IgG2, IgG3, and IgG4), andIgM. The distinguishing feature between these antibody classes is theirconstant regions, although subtler differences may exist in the Vregion. IgG antibodies are tetrameric proteins composed of two heavychains and two light chains. The IgG heavy chain is composed of fourimmunoglobulin domains linked from N- to C-terminus in the orderVH—CH1-CH2-CH3, referring to the heavy chain variable domain, heavychain constant domain 1, heavy chain constant domain 2, and heavy chainconstant domain 3 respectively (also referred to as VH-Cγ1-Cγ2-Cγ3,referring to the heavy chain variable domain, constant gamma 1 domain,constant gamma 2 domain, and constant gamma 3 domain respectively). TheIgG light chain is composed of two immunoglobulin domains linked from N-to C-terminus in the order VL-CL, referring to the light chain variabledomain and the light chain constant domain respectively.

Antibodies have serum half-lives in vivo ranging from one to threeweeks. This favorable property is due to the preclusion of kidneyfiltration due to the large size of the full-length molecule, andinteraction of the antibody Fc region with the neonatal Fc receptorFcRn. Binding to FcRn recycles endocytosed antibody from the endosomeback to the bloodstream (Raghavan et al., 1996, Annu Rev Cell Dev Biol12:181-220; Ghetie et al., 2000, Annu Rev Immunol 18:739-766, bothentirely incorporated by reference).

Other properties of the antibody may determine its clearance rate (e.g.stability and half-life) in vivo. In addition to antibody binding to theFcRn receptor, other factors that contribute to clearance and half-lifeare serum aggregation, enzymatic degradation in the serum, inherentimmunogenicity of the antibody leading to clearing by the immune system,antigen-mediated uptake, FcR (non-FcRn) mediated uptake and non-serumdistribution (e.g. in different tissue compartments).

Recently it has been suggested that antibodies with variable regionsthat have lower isoelectric points may also have longer serum half-lives(Igawa et al., 2010 PEDS. 23(5): 385-392; US Publication 2011/0076275both of which are entirely incorporated by reference). However, themechanism of this is still poorly understood, and in fact the authorssuggest that engineering the variable region is an alternative toengineering the Fc region. Moreover, variable regions differ fromantibody to antibody. As such, each variable region must be alteredwithout significantly affecting the binding affinity.

Accordingly, the present application defines the impact of charge stateon antibody pharmacokinetics, and provides novel engineered variants inthe constant regions to improve serum half-life.

BRIEF SUMMARY OF THE INVENTION Problem to be Solved

Accordingly, one problem to be solved is to increase serum half life ofantibodies by altering the constant domains, thus allowing the sameconstant regions to be used with different antigen binding sequences.e.g. the variable regions including the CDRs, and minimizing thepossibility of immunogenic alterations. Thus providing antibodies withconstant region variants with reduced pI and extended half-life providesa more modular approach to improving the pharmacokinetic properties ofantibodies, as described herein. In addition, due to the methodologiesoutlined herein, the possibility of immunogenicity resulting from the pIvariants is significantly reduced by importing pI variants fromdifferent IgG isotypes such that pI is reduced without introducingsignificant immunogenicity. Thus, an additional problem to be solved isthe elucidation of low pI constant domains with high human sequencecontent, e.g. the minimization or avoidance of non-human residues at anyparticular position.

SUMMARY

Accordingly, one aspect the invention provides methods for modifying theisoelectric point of an antibody by introducing at least 6 amino acidmutations, including substitutions with non-native amino acids in aconstant domain selected from the heavy chain constant domain and lightchain constant domain, wherein the substituted amino acids have a pIlower than the native amino acid, such that said isoelectric point ofthe variant antibody is lowered by at least 0.5 logs. In some cases,only the heavy chain constant domain is altered; in some cases, only thelight chain constant domain, and in some cases both the heavy and lightconstant domains comprise mutated amino acids.

In another aspect the methods provide for the generation of thesevariants by amino acid mutations selected from the group consisting of anon-native glutamic acid at position 119; a non-native cysteine atposition 131; a non-native arginine, lysine or glutamine at position133; a non-native glutamic acid at position 137; a non-native serine atposition 138; a non-native glutamic acid at position 164; a non nativeasparagine at position 192; a non native phenylalanine at position 193,a non-native lysine at position 196, a non-native threonine at position199, a non-native aspartic acid at position 203, a non-native glutamicacid or glutamine at position 205, a non native aspartic acid atposition 208, a non-native glutamic acid or glutamine at position 210, anon native threonine at position 214, a non native arginine at position217 and a non-native cysteine at position 219, a deletion at position221, a non-native valine or threonine at position 222, a deletion atposition 223, a non-native glutamic acid at position 224, a deletion atposition 225, a deletion at position 235, a non-native glutamine orglutamic acid at position 274, a non-native phenylalanine at position296, a non native phenylalanine at position 300, a non-native valine atposition 309, a non-native glutamic acid at position 320, a non-nativeglutamic acid at position 322, a non-native glutamic acid at position326, a non-native glycine at position 327, a non-native glutamic acid atposition 334, a non native threonine at position 339, a non nativeglutamine or glutamic acid at position 355, a non-native serine atposition 384, a non-native asparagine or glutamic acid at position 392,a non-native methionine at position 397, a non native glutamic acid atposition 419, and a deletion or non-native aspartic acid at position447, using EU numbering.

In a further aspect, the invention provides methods for modifying theisoelectric point of an antibody by introducing at least 2 amino acidmutations in the light constant domain, such that said isoelectric pointof the variant antibody is lowered by at least 0.5 logs, and whereinsaid variant antibody comprises substitutions selected from the groupconsisting of a non-native glutamine or glutamic acid at position 126, anon-native glutamine, glutamic acid or threonine at position 145; anon-native aspartic acid at position 152, a non-native glutamic acid atposition 156, a non-native glutamine or glutamic acid at position 169, anon-native glutamic acid at position 199, a non-native glutamic acid atposition 202 and a a non-native glutamic acid at position 207 (using EUnumbering).

In additional aspects, the invention provides methods for modifying theisoelectric point of an antibody by introducing: a) at least 6 aminoacid mutations in the heavy constant domain, wherein said variantantibody comprises mutations selected from the group consisting of anon-native glutamic acid at position 119; a non-native cysteine atposition 131; a non-native arginine, lysine or glutamine at position133; a non-native glutamic acid at position 137; a non-native serine atposition 138; a non-native glutamic acid at position 164; a non nativeasparagine at position 192; a non native phenylalanine at position 193,a non-native lysine at position 196, a non-native threonine at position199, a non-native aspartic acid at position 203, a non-native glutamicacid or glutamine at position 205, a non native aspartic acid atposition 208, a non-native glutamic acid or glutamine at position 210, anon native threonine at position 214, a non native arginine at position217 and a non-native cysteine at position 219, a deletion at position221, a non-native valine or threonine at position 222, a deletion atposition 223, a non-native glutamic acid at position 224, a deletion atposition 225, a deletion at position 235, a non-native glutamine orglutamic acid at position 274, a non-native phenylalanine at position296, a non native phenylalanine at position 300, a non-native valine atposition 309, a non-native glutamic acid at position 320, a non-nativeglutamic acid at position 322, a non-native glutamic acid at position326, a non-native glycine at position 327, a non-native glutamic acid atposition 334, a non native threonine at position 339, a non nativeglutamine or glutamic acid at position 355, a non-native serine atposition 384, a non-native asparagine or glutamic acid at position 392,a non-native methionine at position 397, a non native glutamic acid atposition 419, and a deletion or non-native aspartic acid at position447; and b) substituting at least 2 non-native amino acids in the lightconstant domain, wherein said variant antibody comprises substitutionsselected from the group consisting of a non-native glutamine or glutamicacid at position 126, a non-native glutamine, glutamic acid or threonineat position 145; a non-native aspartic acid at position 152, anon-native glutamic acid at position 156, a non-native glutamine orglutamic acid at position 169, a non-native glutamic acid at position199, a non-native glutamic acid at position 202 and a a non-nativeglutamic acid at position 207 (using EU numbering), such that saidisoelectric point of the variant antibody is lowered by at least 0.5logs.

In a further aspect, the pI antibodies of the invention, generated usingthe above methods, has an increased serum half life as compared to anantibody without the mutations.

In an additional aspect, the invention provides antibodies comprising avariant heavy constant domain polypeptide comprising a variant of SEQ IDNO: 2, comprising at least 6 mutations selected from the groupconsisting of a non-native glutamic acid at position 119; a non-nativecysteine at position 131; a non-native arginine, lysine or glutamine atposition 133; a non-native glutamic acid at position 137; a non-nativeserine at position 138; a non-native glutamic acid at position 164; anon native asparagine at position 192; a non native phenylalanine atposition 193, a non-native lysine at position 196, a non-nativethreonine at position 199, a non-native aspartic acid at position 203, anon-native glutamic acid or glutamine at position 205, a non nativeaspartic acid at position 208, a non-native glutamic acid or glutamineat position 210, a non native threonine at position 214, a non nativearginine at position 217 and a non-native cysteine at position 219, adeletion at position 221, a non-native valine or threonine at position222, a deletion at position 223, a non-native glutamic acid at position224, a deletion at position 225, a deletion at position 235, anon-native glutamine or glutamic acid at position 274, a non-nativephenylalanine at position 296, a non native phenylalanine at position300, a non-native valine at position 309, a non-native glutamic acid atposition 320, a non-native glutamic acid at position 322, a non-nativeglutamic acid at position 326, a non-native glycine at position 327, anon-native glutamic acid at position 334, a non native threonine atposition 339, a non native glutamine or glutamic acid at position 355, anon-native serine at position 384, a non-native asparagine or glutamicacid at position 392, a non-native methionine at position 397, a nonnative glutamic acid at position 419, and a deletion or non-nativeaspartic acid at position 447.

In an additional aspect, the invention provides antibodies comprising avariant light constant domain polypeptide comprising of variant of SEQID NO: 112, wherein said variant antibody comprises substitutionsselected from the group consisting of a non-native glutamine or glutamicacid at position 126, a non-native glutamine, glutamic acid or threonineat position 145; a non-native aspartic acid at position 152, anon-native glutamic acid at position 156, a non-native glutamine orglutamic acid at position 169, a non-native glutamic acid at position199, a non-native glutamic acid at position 202 and a a non-nativeglutamic acid at position 207 (using EU numbering).

In a further aspect, the invention provides nucleic acids encoding theantibodies, including a nucleic acid encoding a variant heavy chainconstant domain and/or a nucleic acid encoding a variant light chainconstant domain. Host cells containing the nucleic acids and methods ofproducing the antibodies are also included.

In an additional aspect, the invention provides antibodies comprising avariant heavy chain constant domain having the formula:

A-X₁₁₉-T-K-G-P-S-V-F-P-L-A-P-X₁₃₁-S-X₁₃₃-S-T-S-X₁₃₇-X₁₃₈-T-A-A-L-G-C-L-V-K-D-Y-F-P-E-P-V-T-V-S-W-N-S-G-A-L-X₁₆₄-S-G-V-H-T-F-P-A-V-L-Q-S-S-G-L-Y-S-L-S-S-V-V-T-V-P-S-S-X₁₉₂-X₁₉₃-G-T-X₁₉₆-T-Y-X₁₉₉-C-N-V-X₂₀₃-H-X₂₀₅-P-S-X₂₀₈-T-X₂₁₀-V-D-K-X₂₁₄-V-E-X₂₁₇-K-X₂₁₉-C-X₂₂₁-X₂₂₂-X₂₂₃-X₂₂₄-X₂₂₅-C-P-P-C-P-A-P-X₂₃₃-X₂₃₄-X₂₃₅-X₂₃₆-G-P-S-V-F-L-F-P-P-K-P-K-D-T-L-M-I-S-R-T-P-E-V-T-C-V-V-V-D-V-S-H-E-D-P-E-V-X₂₇₄-F-N-W-Y-V-D-G-V-E-V-H-N-A-K-T-K-P-R-E-E-Q-X₂₉₆-N-S-T-X₃₀₀-R-V-V-S-V-L-T-V-X₃₀₉-H-Q-D-W-L-N-G-K-E-Y-X₃₂₀-C-X₃₂₂-V-S-N-X₃₂₆-X₃₂₇-L-P-A-P-I-E-X₃₃₄-T-I-S-K-X₃₃₉-K-G-Q-P-R-E-P-Q-V-Y-T-L-P-P-S-X₃₅₅-E-E-M-T-K-N-Q-V-S-L-T-C-L-V-K-G-F-Y-P-S-D-I-A-V-E-W-E-S-X₃₈₄-G-Q-P-E-N-N-Y-X₃₉₂-T-T-P-P-X₃₉₇-L-D-S-D-G-S-F-F-L-Y-S-K-L-T-V-D-K-S-R-W-Q-X₄₁₉-G-N-V-F-S-C-S-V-X₄₂₈-H-E-A-L-H-X₄₃₄-H-Y-T-Q-K-S-L-SL-S-P-G-X₄₄₇,

wherein X₁₁₉ is selected from the group consisting of S and E;

wherein X₁₃₁ is selected from the group consisting of S and C;

wherein X₁₃₃ is selected from the group consisting of K, R, E, and Q;

wherein X₁₃₇ is selected from the group consisting of G and E;

wherein X₁₃₈ is selected from the group consisting of G and S;

wherein X₁₆₄ is selected from the group consisting of T and E;

wherein X₁₉₂ is selected from the group consisting of S and N;

wherein X₁₉₃ is selected from the group consisting of L and F;

wherein X₁₉₆ is selected from the group consisting of Q and K;

wherein X₁₉₉ is selected from the group consisting of I and T;

wherein X₂₀₃ is selected from the group consisting of N and D;

wherein X₂₀₅ is selected from the group consisting of K, E, and Q;

wherein X₂₀₈ is selected from the group consisting of N and D;

wherein X₂₁₀ is selected from the group consisting of K, E, and Q;

wherein X₂₁₄ is selected from the group consisting of K and T;

wherein X₂₁₇ is selected from the group consisting of P and R;

wherein X₂₁₉ is selected from the group consisting of S and C;

wherein X₂₂₀ is selected from the group consisting of C, PLG, and G;

wherein X₂₂₁ is selected from the group consisting of D and a deletion;

wherein X₂₂₂ is selected from the group consisting of K, V, and T;

wherein X₂₂₃ is selected from the group consisting of T and a deletion;

wherein X₂₂₄ is selected from the group consisting of H and E;

wherein X₂₂₅ is selected from the group consisting of T and a deletion;

wherein X₂₃₃ is selected from the group consisting of E and P;

wherein X₂₃₄ is selected from the group consisting of L and V;

wherein X₂₃₅ is selected from the group consisting of L, A, and adeletion;

wherein X₂₃₆ is selected from the group consisting of G, A, and adeletion;

wherein X₂₇₄ is selected from the group consisting of K, Q, and E;

wherein X₂₉₆ is selected from the group consisting of Y and F;

wherein X₃₀₀ is selected from the group consisting of Y and F;

wherein X₃₀₉ is selected from the group consisting of L and V;

wherein X₃₂₀ is selected from the group consisting of K and E;

wherein X₃₂₂ is selected from the group consisting of K and E;

wherein X₃₂₆ is selected from the group consisting of K and E;

wherein X₃₂₇ is selected from the group consisting of A and G;

wherein X₃₃₄ is selected from the group consisting of K and E;

wherein X₃₃₉ is selected from the group consisting of A and T;

wherein X₃₅₅ is selected from the group consisting of R, Q, and E;

wherein X₃₅₄ is selected from the group consisting of N and S;

wherein X₃₉₂ is selected from the group consisting of K, N, and E;

wherein X₃₉₇ is selected from the group consisting of V and M;

wherein X₄₁₉ is selected from the group consisting of Q and E;

wherein X₄₂₈ is selected from the group consisting of M and L;

wherein X₄₃₄ is selected from the group consisting of N and S; and

wherein X₄₄₇-X₄₅₁ is selected from the group consisting of K, DEDE, anda deletion;

wherein said variant heavy chain constant domain comprises at least 6substitutions as compared to SEQ ID NO: 2 and said variant is not SEQ IDNO: 3.

In a further aspect the invention provides variant heavy chain constantdomain comprises at least 10 or 15 substitutions as compared to SEQ IDNO: 2.

In an additional aspect, the invention provides antibodies with avariant light chain constant domain having the formula:

X₁₀₈-T-V-A-A-P-S-V-F-I-F-P-P-S-D-E-X₁₂₄-L-X₁₂₆-S-G-T-A-S-V-V-C-L-L-N-X₁₃₈-F-Y-P-R-E-A-X₁₄₅-V-Q-W-K-V-D-X₁₅₂-A-L-Q-X₁₅₆-G-N-S-Q-E-S-V-T-E-Q-D-S-X₁₆₉-D-S-T-Y-S-L-S-S-T-L-T-L-S-K-A-D-Y-E-K-H-K-V-Y-A-C-E-V-T-H-X₁₉₉-G-L-X₂₀₂-S-P-V-T-X₂₀₇-S-E-N-R-G-E-X₂₁₄,

wherein X₁₀₈ is selected from the group consisting of R and Q;

wherein X₁₂₄ is selected from the group consisting of Q and E;

wherein X₁₂₆ is selected from the group consisting of K, E, and Q;

wherein X₁₃₈ is selected from the group consisting of N and D;

wherein X₁₄₅ is selected from the group consisting of K, E, Q, and T;

wherein X₁₅₂ is selected from the group consisting of N and D;

wherein X₁₅₆ is selected from the group consisting of S and E;

wherein X₁₆₉ is selected from the group consisting of K, E, and Q;

wherein X₁₉₉ is selected from the group consisting of Q and E;

wherein X₂₀₂ is selected from the group consisting of S and E; and

wherein X₂₀₇ is selected from the group consisting of K and E; and

wherein X₂₁₄-X₂₁₈ is selected from the group consisting of C and CDEDE.

wherein said variant light chain constant domain comprises at least 2substitutions as compared to SEQ ID NO: 112.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequences of wild-type constant regions used in theinvention, [SEQ ID NOs. 1-6].

FIG. 2. Engineering of heavy chain CH1 domains. List of CH1 residues forthe four IgG isotypes, fraction exposed, and examples of substitutionsthat can be made to lower pI. Numbering is according to the EU index.

FIG. 3. Engineering of light chain CK domains. List of CK residues,fraction exposed, and substitutions that can be made to lower pI.Numbering is according to the EU index.

FIG. 4. Amino acid sequences of pI engineered constant regionsIgG1-CH1-pI(6) and CK-pI(6) [SEQ ID NOs. 7-8, 52-53].

FIG. 5. Amino acid sequences of wild-type anti-VEGF VH and VL variableregions used in the invention [SEQ ID NOs. 9-10].

FIG. 6. Amino acid sequences of the heavy and light chains of pIengineered anti-VEGF antibody XENP9493 IgG1-CH1-pI(6)-CK-pI(6) used inthe invention [SEQ ID NOs. 11-12].

FIG. 7. Structure of an antibody Fab domain showing the locations of pIlowering mutations in XENP9493 IgG1-CH1-pI(6)-CK-pI(6).

FIG. 8. Analysis of pI engineered anti-VEGF variants on an AgilentBioanalyzer showing high purity.

FIG. 9. Analysis of pI engineered anti-VEGF variants on SEC showing highpurity.

FIG. 10. Analysis of pI engineered anti-VEGF variants on an IEF gelshowing variants have altered pI.

FIG. 11. Binding analysis (Biacore) of bevacizumab and pI engineeredanti-VEGF binding to VEGF.

FIG. 12. DSC analysis of CH1 and CK pI engineered anti-VEGF showing highthermostability.

FIG. 13. PK of bevacizumab variants in huFcRn mice. The 9493 variantwith pI-engineered CH1 and CK domains extends half-life in vivo.

FIG. 14. PK of a native IgG1 version of bevacizumab in four separate invivo studies in huFcRn mice. The average IgG1 half-life was 3.2 days.

FIG. 15. PK of a native IgG2 version of bevacizumab in huFcRn mice.

FIG. 16. Correlation between half-life and isoelectric point (pI) ofantibody variants with different constant chains.

FIG. 17. Amino acid sequence alignment of the IgG subclasses. Residueswith a bounded box illustrate isotypic differences between the IgG's.Residues which contribute to a higher pI (K, R, and H) or lower pI (Dand E) are highlighted in bold. Designed substitutions that either lowerthe pI, or extend an epitope are shown in gray [SEQ ID NOs. 2-5].

FIG. 18. Amino acid sequence of the CK and Cλ light constant chains.Residues which contribute to a higher pI (K, R, and H) or lower pI (Dand E) are highlighted in bold. Preferred positions that can be modifiedto lower the pI are shown in gray [SEQ ID NOs. 208-209].

FIG. 19. Amino acid sequences of pI-engineered variant heavy chains[SEQ. ID NOs. 2.3, 13-16, 19, 50-51, 55-57].

FIG. 20. Amino acid sequences of pI-engineered variant light chains [1,17, 8, 18, 10, 54, 58, 59-60].

FIG. 21. PK results of pI-engineered variant bevacizumab antibodies inhuFcRn mice.

FIG. 22. PK results of variants that combine pI-engineered modificationswith Fc modifications that enhance binding to FcRn.

FIG. 23. Correlation between half-life and isoelectric point (pI) ofnative bevacizumab antibodies, pI-engineered variant versions withreduced pI, and native and pI-engineered versions that incorporate Fcmodifications that improve binding to human FcRn.

FIG. 24. Amino acid sequence alignment of novel isotype IgG-pI-Iso3 withthe IgG subclasses. Blue indicates a match between pI-iso3 and residuesin the four native IgG's IgG1, IgG2, IgG3, and IgG4. Residues with abounded box illustrate IgG isotypic differences that have beenincorporated into IgG-pI-Iso3 that reduce pI [SEQ ID NOs. 2-5].

FIG. 25. Differences between IgG and IgG-pI-Iso3 in the hinge and Fcregion [SEQ ID NOs. 210-211].

FIG. 26. Differences between IgG1 and IgG-pI-Iso3 in the CH1 region.

FIG. 27. Amino acid illustration of the CK-pI(4) variant. Red indicateslysine to glutamic acid charge substitutions relative to the native CKlight constant chain [SEQ ID NOs. 212].

FIG. 28. Amino acid sequences of pI-engineered heavy and light constantchains [SEQ ID NOs. 19, 61, 65, 20-21, 67, 22, 62-64, 23-24, 66, 25, 68,26-27, 69, 28-33].

FIG. 29. Analysis of basic residues in the antibody Fc region showingfraction exposed and the calculated energy for substitution to Glunormalized against the energy of the WT residue. Basic residues with ahigh fraction exposed and a favorable delta E for substitution to Gluare targets for charge swap mutations to lower pI.

FIG. 30. Plot showing the effect of charge swap mutations on antibodypI. As the pI gets lower the change in pI per charge swap decreases.

FIG. 31. PK results of pI-engineered isotypic variant bevacizumabantibodies (IgG-pI-Iso3) and combinations with substitution N434S inhuFcRn mice.

FIG. 32. PK results of pI-engineered isotypic variant bevacizumabantibodies and combinations with substitution N434S in huFcRn mice.

FIG. 33. Scatter plot of PK results of pI-engineered isotypic variantbevacizumab antibodies and combinations with substitution N434S inhuFcRn mice. Each point represents a single mouse from the study. Itshould be noted that the 428L substitution can also be added to each ofthese pI antibodies.

FIG. 34. Plot showing correlation between pI engineered variant pI andhalf-life (t½).

FIG. 35. Structural alignment of CK and C-lambda domains.

FIG. 36. Literature pIs of the 20 amino acids. It should be noted thatthe listed pIs are calculated as free amino acids; the actual pI of anyside chain in the context of a protein is different, and thus this listis used to show pI trends and not absolute numbers for the purposes ofthe invention.

FIGS. 37A-37F. Data table of exemplary pI-engineered variants listing:

XenP# the internal reference number Name (HC) heavy chain sequencedesignation SEQ ID NO (HC) corresponding SEQ ID NO of the heavy chainsequence Name (LC) light chain sequence designation SEQ ID NO (LC)corresponding SEQ ID NO of the light chain sequence Calc. pI calculatedpI value for the entire antibody sequence, including heavy and lightchain Fv + constant domains, with the Fv of bevacizumab and the constantdomains as defined in the table #KR number of Lys or Arg residues inIgG1 with the Fv of bevacizumab and the constant domains as defined inthe table Delta KR change in the number of Lys or Arg residues relativeto (vs. WT) IgG1 wild-type sequence of bevacizumab #DE number of Asp orGlu residues in IgG1 with the Fv of bevacizumab and the constant domainsas defined in the table Delta DE change in the number of Asp or Glu acidresidues (vs. WT) relative to IgG1 wild-type sequence of bevacizumabCharge state derived from the total number of Lys and Arg minus thetotal number of Asp and Glu residues, assuming a pH of 7 # HC Mutationsnumber of mutations in the heavy chain constant vs IgG1 domain ascompared to IgG1 # LC Mutations number of mutations in the light chainconstant vs IgG1 domain as compared to IgG1 Total # of total number ofmutations in the heavy chain and light Mutations chain constant domainsas compared to IgG1

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The present invention is generally directed to compositions and methodsrelating to decreasing the isoelectric point (pI) of antibodies (to form“pI antibodies”) by incorporating amino acid substitutions (“pIvariants” or “pI substitutions”) into one or more constant regiondomains of the antibody. The pI substitutions are chosen such that thepI amino acids have a pI lower than the native amino acid at aparticular position in the constant domain. In various embodiments, theconstant domain variants reduce the pI of the antibody, and, as shownherein for the first time, improve serum half-life in vivo. While, asnoted above, there is limited data that might suggest that lowering thepI of an antibody by generating variants in the CDR regions of anantibody can lead to increased serum half life. However, the presentinvention provides a significant benefit to CDR pI engineering, as theconstant domains of the present invention can be added in a modularfashion to the variable regions, thus significantly simplifying designof antibodies that have increased serum half lives.

That is, until the present invention, the fact that decreasing pI of anantibody would lead to increased serum half life was both unpredictableand unexpected.

In addition, many embodiments of the invention rely on the “importation”of lower pI amino acids at particular positions from one IgG isotypeinto another, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. That is, IgG1 is acommon isotype for therapeutic antibodies for a variety of reasons,including high effector function. However, the heavy constant region ofIgG1 has a higher pI than that of IgG2 (8.10 versus 7.31). Byintroducing IgG2 residues at particular positions into the IgG1backbone, the pI of the resulting protein is lowered, and additionallyexhibits longer serum half-life. For example, IgG1 has a glycine (pI5.97) at position 137, and IgG2 has a glutamic acid (pI 3.22); importingthe glutamic acid will affect the pI of the resulting protein. As isdescribed below, a number of amino acid substitutions are generallyrequired to significant affect the pI of the variant antibody. However,it should be noted as discussed below that even changes in IgG2molecules allow for increased serum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g. bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant decreases in pI of the resulting antibody can beseen. As discussed below, lowering the pI by at least 0.5 can increasethe half life significantly.

As will be appreciated by those in the art and described below, a numberof factors contribute to the in vivo clearance, and thus the half-life,of antibodies in serum. One factor involves the antigen to which theantibody binds; that is, antibodies with identical constant regions butdifferent variable regions (e.g. Fv domains), may have differenthalf-lives due to differential ligand binding effects. However, thepresent invention demonstrates that while the absolute half life of twodifferent antibodies may differ due to these antigen specificityeffects, the pI variants (which optionally include FcRn variants asoutlined herein), can transfer to different ligands to give the sametrends of increasing half-life. That is, in general, the relative“order” of the pI decreases/half life increases will track to antibodieswith the same pI variants of antibodies with different Fvs as isdiscussed herein.

II. Description of the Invention

A. Antibodies

The present invention relates to the generation of pI variants ofantibodies, generally therapeutic antibodies. As is discussed below, theterm “antibody” is used generally. Antibodies that find use in thepresent invention can take on a number of formats as described herein,including traditional antibodies as well as antibody derivatives,fragments and mimetics, described below. In general, the term “antibody”includes any polypeptide that includes at least one constant domain,including, but not limited to, CH1, CH2, CH3 and CL.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to the IgG class, which has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4. Thus, “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Itshould be understood that therapeutic antibodies can also comprisehybrids of isotypes and/or subclasses. For example, as shown herein, thepresent invention covers pI engineering of IgG1/G2 hybrids.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5^(th) Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) (e.g. Kabat et al.,supra (1991)).

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.”

As will be appreciated by those in the art, a wide variant of antigenbinding domains, e.g. Fv regions, may find use in the present invention.Virtually any antigen may be targeted by the IgG variants, including butnot limited to proteins, subunits, domains, motifs, and/or epitopesbelonging to the following list of target antigens, which includes bothsoluble factors such as cytokines and membrane-bound factors, includingtransmembrane receptors: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a,8-oxo-dG, A1 Adenosine Receptor. A33, ACE, ACE-2, Activin, Activin A,Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2,Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12,ADAM15, ADAM17/TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins,aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART,Artemin, anti-Id, ASPARTIC, Atrial natriuretic factor, av/b3 integrin,Ax1, b2M, B7-1, B7-2, B7-H, B-lymphocyte Stimulator (BlyS), BACE,BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA,BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a,BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8(BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1,BMPR-II (BRK-3), BMPs, b-NGF, BOK, Bombesin, Bone-derived neurotrophicfactor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a,C10, CA 125, CAD-8, Calcitonin, cAMP, carcinoembryonic antigen (CEA),carcinoma-associated antigen, Cathepsin A, Cathepsin B, CathepsinC/DPPI, Cathepsin D. Cathepsin E, Cathepsin H. Cathepsin L, Cathepsin O,Cathepsin S, Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1,CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2,CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3.CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5,CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD3, CD4, CD15, CD16, CD15,CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L,CD32, CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46,CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1),CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152,CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin,Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1,COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL,CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10,CXCL11, CXCL12, CXCL13, CXCL14, CXCL5, CXCL16, CXCR, CXCR1, CXCR2,CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN,DCC, DcR3, DC-SIGN, Decay accelerating factor, des(1-3)-IGF-I (brainIGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA,EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelinreceptor, Enkephalinase, eNOS, Eot, eotaxin1, EpCAM, Ephrin B2/EphB4,EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc,Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1,Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL,FLIP, Flt-3, Flt-4, Follicle stimulating hormone, Fractalkine, FZD1,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6,GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14,CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8(Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1,GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut 4, glycoproteinIIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growth hormonereleasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBenvelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma. IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5. Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP, LDGF,LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3,Lfo, LIF, LIGHT, lipoproteins. LIX, LKN, Lptn, L-Selectin, LT-a, LT-b,LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin BetaReceptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF,MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG,MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Muc1), MUC18, Muellerian-inhibitin substance, Mug,MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN,OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP,PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4,PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP),PIGF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA,prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51,RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R Apo-2, DR4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95). TNFRSF6B (DcR3 M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25(DR3 Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand,TL2), TNFSF11 (TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL A/VEGI),TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate. TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (fit-1), VEGF, VEGFR, VEGFR-3(fit-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors.

In some embodiments, the pI engineering described herein is done totherapeutic antibodies. A number of antibodies that are approved foruse, in clinical trials, or in development may benefit from the pIvariants of the present invention. These antibodies are herein referredto as “clinical products and candidates”. Thus in a preferredembodiment, the pI engineered constant region(s) of the presentinvention may find use in a range of clinical products and candidates.For example, a number of antibodies that target CD20 may benefit fromthe pI engineering of the present invention. For example the pI variantsof the present invention may find use in an antibody that issubstantially similar to rituximab (Rituxan®, IDEC/Genentech/Roche) (seefor example U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibodyapproved to treat Non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20currently being developed by Genmab, an anti-CD20 antibody described inU.S. Pat. No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20(Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769(PCT/US2003/040426, entitled “Immunoglobulin Variants and UsesThereof”). A number of antibodies that target members of the family ofepidermal growth factor receptors, including EGFR (ErbB-1), Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), may benefit from pI engineeredconstant region(s) of the invention. For example the pI engineeredconstant region(s) of the invention may find use in an antibody that issubstantially similar to trastuzumab (Herceptin®, Genentech) (see forexample U.S. Pat. No. 5,677,171), a humanized anti-Her2neu antibodyapproved to treat breast cancer, pertuzumab (rhuMab-2C4, Omnitarg™),currently being developed by Genentech; an anti-Her2 antibody describedin U.S. Pat. No. 4,753,894; cetuximab (Erbitux®, Imclone) (U.S. Pat. No.4,943,533; PCT WO 96/40210), a chimeric anti-EGFR antibody in clinicaltrials for a variety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883),currently being developed by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S.Ser. No. 10/172,317), currently being developed by Genmab; 425,EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864;Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al.,1987, J Cell Biochem. 35(4):315-20; Kettleborough et al., 1991, ProteinEng. 4(7):773-83); ICR62 (Institute of Cancer Research) (PCT WO95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993,22(1-3):129-46; Modjtahedi et al., 1993, Br J Cancer. 1993,67(2):247-53; Modjtahcdi et al, 1996, Br J Cancer, 73(2):228-35;Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YMBiosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S. Pat.No. 5,891,996; U.S. Pat. No. 6,506,883; Mateo et al, 1997,Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Institute for CancerResearch, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc NatlAcad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (IVAX,National Cancer Institute) (PCT WO 0162931A2); and SC100 (Scancell) (PCTWO 01/88138). In another preferred embodiment, the pI engineeredconstant region(s) of the present invention may find use in alemtuzumab(Campath®, Millenium), a humanized monoclonal antibody currentlyapproved for treatment of B-cell chronic lymphocytic leukemia. The pIengineered constant region(s) of the present invention may find use in avariety of antibodies that are substantially similar to other clinicalproducts and candidates, including but not limited to muromonab-CD3(Orthoclone OKT3®), an anti-CD3 antibody developed by OrthoBiotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), an anti-CD20antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin(Mylotarg®), an anti-CD33 (p67 protein) antibody developed byCelltech/Wyeth, alefacept (Amevive®)), an anti-LFA-3 Fc fusion developedby Biogen), abciximab (ReoPro®), developed by Centocor/Lilly,basiliximab (Simulect®), developed by Novartis, palivizumab (Synagis®),developed by MedImmune, infliximab (Remicadei®), an anti-TNFalphaantibody developed by Centocor, adalimumab (Humira®)), an anti-TNFalphaantibody developed by Abbott, Humicade™, an anti-TNFalpha antibodydeveloped by Celltech, etanercept (Enbrel®), an anti-TNFalpha Fc fusiondeveloped by Immunex/Amgen, ABX-CBL, an anti-CD147 antibody beingdeveloped by Abgenix, ABX-IL8, an anti-IL8 antibody being developed byAbgenix, ABX-MA1, an anti-MUC18 antibody being developed by Abgenix,Pemtumomab (R1549, 90Y-muHMFG 1), an anti-MUC1 In development byAntisoma, Therex (R1550), an anti-MUC1 antibody being developed byAntisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, beingdeveloped by Antisoma. Thioplatin (AS1407) being developed by Antisoma,Antegren®) (natalizumab), an anti-alpha-4-beta-1 (VLA-4) andalpha-4-beta-7 antibody being developed by Biogen. VLA-1 mAb, ananti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, ananti-lymphotoxin beta receptor (LTBR) antibody being developed byBiogen, CAT-152, an anti-TGF-32 antibody being developed by CambridgeAntibody Technology, J695, an anti-IL-12 antibody being developed byCambridge Antibody Technology and Abbott, CAT-192, an anti-TGFβ1antibody being developed by Cambridge Antibody Technology and Genzyme,CAT-213, an anti-Eotaxin1 antibody being developed by Cambridge AntibodyTechnology, LymphoStat-B™ an anti-Blys antibody being developed byCambridge Antibody Technology and Human Genome Sciences Inc.,TRAIL-R1mAb, an anti-TRAIL-R1 antibody being developed by CambridgeAntibody Technology and Human Genome Sciences, Inc., Avastin™(bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed byGenentech, an anti-HER receptor family antibody being developed byGenentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibodybeing developed by Genentech, Xolair™ (Omalizumab), an anti-IgE antibodybeing developed by Genentech, Raptiva™ (Efalizumab), an anti-CD11aantibody being developed by Genentech and Xoma, MLN-02 Antibody(formerly LDP-02), being developed by Genentech and MilleniumPharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed byGenmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab andAmgen, HuMax-Inflam, being developed by Genmab and Medarex,HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmaband Medarex and Oxford GeoSciences, HuMax-Lymphoma, being developed byGenmab and Amgen, HuMax-TAC, being developed by Genmab. IDEC-131, andanti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151(Clenoliximab), an anti-CD4 antibody being developed by IDECPharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDECPharmaceuticals, IDEC-152, an anti-CD23 being developed by IDECPharmaceuticals, anti-macrophage migration factor (MIF) antibodies beingdeveloped by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibodybeing developed by Imclone. IMC-1C11, an anti-KDR antibody beingdeveloped by Imclone, DC101, an anti-flk-1 antibody being developed byImclone, anti-VE cadherin antibodies being developed by Imclone,CEA-Cide™ (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibodybeing developed by Immunomedics, LymphoCide™ (Epratuzumab), an anti-CD22antibody being developed by Immunomedics, AFP-Cide, being developed byImmunomedics, MyelomaCide, being developed by Immunomedics, LkoCide,being developed by Immunomedics, ProstaCide, being developed byImmunomedics, MDX-010, an anti-CTLA4 antibody being developed byMedarex, MDX-060, an anti-CD30 antibody being developed by Medarex,MDX-070 being developed by Medarex, MDX-018 being developed by Medarex,Osidem™ (IDM-1), and anti-Her2 antibody being developed by Medarex andImmuno-Designed Molecules, HuMax™-CD4, an anti-CD4 antibody beingdeveloped by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody beingdeveloped by Medarex and Genmab, CNTO 148, an anti-TNFα antibody beingdeveloped by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAF™, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-α5β1 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-Ep-CAM antibody being developed by Xoma, and MLN01, ananti-Beta2 integrin antibody being developed by Xoma, an pI-ADC antibodybeing developed by Seattle Genetics, all of the above-cited referencesin this paragraph are expressly incorporated herein by reference.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5^(th)edition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

It should be noted that the sequences depicted herein start at the CH1region, position 118; the variable regions are not included except asnoted. For example, the first amino acid of SEQ ID NO: 2, whiledesignated as position “1” in the sequence listing, corresponds toposition 118 of the CH1 region, according to EU numbering.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230. As noted herein, pI variants can bemade in the hinge region as well.

The light chain generally comprises two domains, the variable lightdomain (containing the light chain CDRs and together with the variableheavy domains forming the Fv region), and a constant light chain region(often referred to as CL or Cκ).

Another region of interest for additional substitutions, outlined below,is the Fc region. By “Fc” or “Fc region” or “Fc domain” as used hereinis meant the polypeptide comprising the constant region of an antibodyexcluding the first constant region immunoglobulin domain and in somecases, part of the hinge. Thus Fc refers to the last two constant regionimmunoglobulin domains of IgA, IgD, and IgG, the last three constantregion immunoglobulin domains of IgE and IgM, and the flexible hingeN-terminal to these domains. For IgA and IgM, Fc may include the Jchain. For IgG, the Fc domain comprises immunoglobulin domains Cγ2 andCγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2(Cγ2). Although the boundaries of the Fc region may vary, the human IgGheavy chain Fc region is usually defined to include residues C226 orP230 to its carboxyl-terminus, wherein the numbering is according to theEU index as in Kabat. In some embodiments, as is more fully describedbelow, amino acid modifications are made to the Fc region, for exampleto alter binding to one or more FcγR receptors or to the FcRn receptor.

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein.

Alternatively, the antibodies can be a variety of structures, including,but not limited to, antibody fragments, monoclonal antibodies,bispecific antibodies, minibodies, domain antibodies, syntheticantibodies (sometimes referred to herein as “antibody mimetics”),chimeric antibodies, humanized antibodies, antibody fusions (sometimesreferred to as “antibody conjugates”), and fragments of each,respectively.

In one embodiment, the antibody is an antibody fragment, as long as itcontains at least one constant domain which can be pI engineered.Specific antibody fragments include, but are not limited to, (i) the Fabfragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al., 1988, Science 242:423-426, Huston etal., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, entirelyincorporated by reference), (iv) “diabodies” or “triabodies”,multivalent or multispecific fragments constructed by gene fusion(Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; WO94/13804;Holliger et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, allentirely incorporated by reference).

Other antibody fragments that can be used include fragments that containone or more of the CH1, CH2, CH3, hinge and CL domains of the inventionthat have been pI engineered. For example, Fc fusions are fusions of theFc region (CH2 and CH3, optionally with the hinge region) fused toanother protein. A number of Fc fusions are known the art and can beimproved by the addition of the pI variants of the invention. In thepresent case, antibody fusions can be made comprising CH1; CH1, CH2 andCH3; CH2; CH3; CH2 and CH3; CH1 and CH3, any or all of which can be madeoptionally with the hinge region, utilizing any combination of pIvariants described herein.

B. Chimeric and Humanized Antibodies

In some embodiments, the antibody can be a mixture from differentspecies, e.g. a chimeric antibody and/or a humanized antibody. Ingeneral, both “chimeric antibodies” and “humanized antibodies” refer toantibodies that combine regions from more than one species. For example,“chimeric antibodies” traditionally comprise variable region(s) from amouse (or rat, in some cases) and the constant region(s) from a human.“Humanized antibodies” generally refer to non-human antibodies that havehad the variable-domain framework regions swapped for sequences found inhuman antibodies. Generally, in a humanized antibody, the entireantibody, except the CDRs, is encoded by a polynucleotide of humanorigin or is identical to such an antibody except within its CDRs. TheCDRs, some or all of which are encoded by nucleic acids originating in anon-human organism, are grafted into the beta-sheet framework of a humanantibody variable region to create an antibody, the specificity of whichis determined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporatedby reference. “Backmutation” of selected acceptor framework residues tothe corresponding donor residues is often required to regain affinitythat is lost in the initial grafted construct (U.S. Pat. No. 5,530,101;U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat.No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213, allentirely incorporated by reference). The humanized antibody optimallyalso will comprise at least a portion of an immunoglobulin constantregion, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. Humanized antibodies can also begenerated using mice with a genetically engineered immune system. Roqueet al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated byreference. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein, all entirely incorporated by reference). Humanizationmethods include but are not limited to methods described in Jones etal., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen etal., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J.Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman etal., 1991. Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al.,1998, Protein Eng 11:321-8, all entirely incorporated by reference.Humanization or other methods of reducing the immunogenicity of nonhumanantibody variable regions may include resurfacing methods, as describedfor example in Roguska et al., 1994. Proc. Natl. Acad. Sci. USA91:969-973, entirely incorporated by reference. In one embodiment, theparent antibody has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, including but not limited to methodsdescribed in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al.,1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol.Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering16(10):753-759, all entirely incorporated by reference. Otherhumanization methods may involve the grafting of only parts of the CDRs,including but not limited to methods described in U.S. Ser. No.09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084, all entirely incorporated byreference.

In one embodiment, the antibodies of the invention can be multispecificantibodies, and notably bispecific antibodies, also sometimes referredto as “diabodies”. These are antibodies that bind to two (or more)different antigens, or different epitopes on the same antigen. Diabodiescan be manufactured in a variety of ways known in the art (Holliger andWinter, 1993, Current Opinion Biotechnol. 4:446-449, entirelyincorporated by reference), e.g., prepared chemically or from hybridhybridomas. In some cases, multispecific (for example bispecific)antibodies are not preferred.

In one embodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scFv joined to a CH3 domain. Hu etal., 1996, Cancer Res. 56:3055-3061, entirely incorporated by reference.In the present instance, the CH3 domain can be pI engineered. In somecases, the scFv can be joined to the Fc region, and may include some orthe entire hinge region.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10⁻⁴ M, at least about 10⁻⁵ M, at least about10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, at least about10⁻⁹ M, alternatively at least about 10⁻¹⁰ M, at least about 10⁻¹¹ M, atleast about 10⁻¹² M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction.

C. pI Variants

The present invention relates to the generation of pI variants ofantibodies. “pI” refers to the isoelectric point of a molecule(including both the individual amino acids and antibodies) and is the pHat which a particular molecule or surface carries no net electricalcharge. In addition, the invention herein sometimes refers to changes inthe “charge state” of the proteins at pH 7. That is, wild-type heavyconstant region of IgG1 has a charge state of +6, while the heavyconstant region of IgG2 has a charge state of 0. Variant 9493 (with aSEQ ID NO: 193 heavy chain constant domain and a SEQ ID NO: 117 lightchain constant domain) has 12 substitutions in both the heavy and lightconstant regions resulting in a charge state of −30.

The present invention relates to the generation of pI variants ofantibodies to form “pI antibodies”. pI variants are made by introducingamino acid mutations into the parent molecule. “Mutations” in thiscontext are usually amino acid substitutions, although as shown herein,deletions and insertions of amino acids can also be done and thus aredefined as mutations.

By “pI variants” or “isoclectric point variants” or “pI substitutions”or grammatical equivalents thereof herein is meant mutating an aminoacid to result in a lower pI at that position. In many embodiments, thismeans making an amino acid substitution with a lower pI than theoriginal (e.g. wild type) amino acid at the particular position. In someembodiments, this can also mean deleting an amino acid with a high pI(if the structure will tolerate it) or inserting amino acids with lowerpIs, for example the low pI “tails” discussed below.

As shown in FIG. 36, the different amino acids have different pIs,although this figure shows the pI of amino acids as individualcompositions rather than in the context of a protein, although the trendis identical. pI variants in the context of the invention are made tocontribute to the decrease of the pI of the protein, in this case atleast the heavy constant domain or the light constant domain of an IgGantibody, or both. An antibody engineered to include one or more of theamino acid mutations outlined herein is sometimes also referred toherein as a “pI antibody”.

In general, “pI variants” refer to the mutation of a higher pI aminoacid either via substituting with an amino acid with a lower pI,deleting an amino acid, or inserting low pI amino acids, thus loweringthe overall pI of the antibody. (As is noted below, additional non-pIvariants are often added to structurally compensate for the pI variants,leading to increased stability, etc.). In the selection of constantdomain positions for alteration with a lower pI amino acid, the solventaccessibility of the amino acid is taken into account, although ingeneral it is not the only factor. That is, based on the known structureof IgG molecules, and as shown in FIG. 2, each position will either befully exposed, fully shielded (e.g. in the interior of the molecule), orpartially exposed. This evaluation is shown in FIG. 2 as a “fractionexposed” of each residue in the CH1 domain and in Cκ light. In someembodiments, candidate positions for substitution with lower pI aminoacids are at least 50% exposed, with exposures of over 60, 70, 80+%finding use in the present invention, as well as those residues that areeffectively 100% exposed.

While not shown, the same calculations can be done for the hinge region,CH2 and CH3 of the heavy chain and the CL domain of the light chain,using standard and commercially available programs to calculate thepercentage exposure.

The lowering of the pI can be done in one of several ways, eitherreplacing a higher pI amino acid (e.g. positive charge state, forexample) with a neutral pI, replacing a higher pI amino acid with alower or low pI amino acid, or replacing a neutral pI amino acid with alow pI amino acid. In some cases, when the structure allows it,deletions or insertions of one or more amino acids can also be done,e.g. deleting a high pI amino acid or inserting one or more low pI aminoacids. Thus, for example, an arginine (pI 11.15) can be replaced bylysine (pI 9.59, still high but lower), a more neutral amino acid likeglycine or serine, or by low pI variants such as aspartic acid orglutamic acid.

pI variants are defined as variants by comparison to the starting orparent sequence, which frequently is the wild-type IgG constant domain(either heavy or light or both, as outlined herein). That is, the aminoacid at a particular position in the wild-type is referred to as the“native” amino acid, and an amino acid substitution (or deletion orinsertion) at this position is referred to as a “non-native” amino acid.For example, many embodiments herein use the IgG1 heavy chain constantregion as a parent sequence in which pI mutations are made. Thus, insome embodiments, a “non-native” amino acid is as compared to the IgG1sequence. For example, at position 119, IgG1 has a serine, and thus thenon-native amino acid that can be substituted is glutamic acid. Thus,SEQ ID NO: 193 has a non-native glutamic acid at position 119.Similarly, when starting with IgG2 constant domain(s), the native andnon-native amino acids are compared to the wild-type IgG2 sequence.

As will be appreciated by those in the art, it is possible to makefusions or hybrids from the various IgG molecules. Thus, for example,SEQ ID NO: 28 is a hybrid IgG1/G2 molecule, and SEQ ID NO: 164 is ahybrid IgG2/G1 molecule. In this context, “non-native” or “non-wildtype” substitutions means that the amino acid at the position inquestion is different from the parent wild-type sequence from whencethat position came; that is, if the cross-over point is between aminoacids 100 and 101, such that the N-terminus is from IgG1 and theC-terminus is from IgG2, a “non-native” amino acid at position 90 willbe compared to the IgG1 sequence.

Thus, it is possible to use non-wild type IgG domains, e.g. IgG domainsthat already have variants, as the starting or parent molecule. In thesecases, as above, a substitution will be “non-native” as long as it doesnot revert back to a wild type sequence.

In general, the pI variants of the invention are chosen to decrease thepositive charge of the pI antibody.

Heavy Chain pI Variants

In some embodiments, the pI variants are made at least in the CH1 regionof the heavy chain domain of an IgG antibody. In this embodiment, themutations can be independently and optionally selected from position119, 131, 133, 137, 138, 164, 192, 193, 196, 199, 203, 205, 208, 210,214, 217 and 219. All possible combinations of these 17 positions can bemade; e.g. a pI antibody may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, or 17 CH1 pI substitutions. In addition, as is describedherein, any single or combination CH1 variant(s) can be combined,optionally and individually, with any CH2, CH3, hinge and LC variant(s)as well, as is further described below.

In addition, the substitution of aspartic acid or glutamic acid atpositions 121, 124, 129, 132, 134, 126, 152, 155, 157, 159, 101, 161,162, 165, 176, 177, 178, 190, 191, 194, 195, 197, 212, 216 and 218 canbe made, as shown in FIG. 2.

Specific substitutions that find use in lowering the pI of CH1 domainsinclude, but are not limited to, a non-native glutamic acid at position119; a non-native cysteine at position 131; a non-native arginine,lysine or glutamine at position 133; a non-native glutamic acid atposition 137; a non-native serine at position 138; a non-native glutamicacid at position 164; a non native asparagine at position 192; a nonnative phenylalanine at position 193, a non-native lysine at position196, a non-native threonine at position 199, a non-native aspartic acidat position 203, a non-native glutamic acid or glutamine at position205, a non native aspartic acid at position 208, a non-native glutamicacid or glutamine at position 210, a non native threonine at position214, a non native arginine at position 217 and a non-native cysteine atposition 219. As is discussed herein, these substitutions can be madeindividually and in any combination, with preferred combinations shownin the SEQ ID listings and described below. In some cases, only pIsubstitutions are done in the CH1 domain, and in others, thesesubstitution(s) are added to other pI variants in other domains in anycombination.

In some embodiments, mutations are made in the hinge domain, includingpositions 221, 222, 223, 224, 225, 233, 234, 235 and 236. It should benoted that changes in 233-236 can be made to increase effector function(along with 327A) in the IgG2 backbone. Thus, pI mutations andparticularly substitutions can be made in one or more of positions221-225, with 1, 2, 3, 4 or 5 mutations finding use in the presentinvention. Again, all possible combinations are contemplated, alone orwith other pI variants in other domains.

Specific substitutions that find use in lowering the pI of hinge domainsinclude, but are not limited to, a deletion at position 221, anon-native valine or threonine at position 222, a deletion at position223, a non-native glutamic acid at position 224, a deletion at position225, a deletion at position 235 and a deletion or a non-native alanineat position 236. Again, as above, these mutations can be madeindividually and in any combination, with preferred combinations shownin the SEQ ID listings and described below. In some cases, only pIsubstitutions are done in the hinge domain, and in others, thesesubstitution(s) are added to other pI variants in other domains in anycombination.

In some embodiments, mutations can be made in the CH2 region, includingpositions 274, 296, 300, 309, 320, 322, 326, 327, 334 and 339. Again,all possible combinations of these 10 positions can be made; e.g. a pIantibody may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 CH2 pI substitutions.

Specific substitutions that find use in lowering the pI of CH2 domainsinclude, but are not limited to, a non-native glutamine or glutamic acidat position 274, a non-native phenylalanine at position 296, a nonnative phenylalanine at position 300, a non-native valine at position309, a non-native glutamic acid at position 320, a non-native glutamicacid at position 322, a non-native glutamic acid at position 326, anon-native glycine at position 327, a non-native glutamic acid atposition 334, a non native threonine at position 339, and all possiblecombinations within CH2 and with other domains.

In this embodiment, the mutations can be independently and optionallyselected from position 355, 384, 392, 397, 419 and 447. All possiblecombinations of these 6 positions can be made; e.g. a pI antibody mayhave 1, 2, 3, 4, 5 or 6 CH1 pI mutations. In addition, as is describedherein, any single or combination CH3 variant(s) can be combined,optionally and individually, with any CH2, CH1, hinge and LC variant(s)as well, as is further described below.

Specific substitutions that find use in lowering the pI of CH3 domainsinclude, but are not limited to, a non native glutamine or glutamic acidat position 355, a non-native serine at position 384, a non-nativeasparagine or glutamic acid at position 392, a non-native methionine atposition 397, a non native glutamic acid at position 419, and a deletionor non-native aspartic acid at position 447.

Thus, taken together, any possible combination of the following heavychain constant domain mutations can be made, with each mutation beingoptionally included or excluded: a non-native glutamic acid at position119; a non-native cysteine at position 131; a non-native arginine,lysine or glutamine at position 133; a non-native glutamic acid atposition 137; a non-native serine at position 138; a non-native glutamicacid at position 164; a non native asparagine at position 192; a nonnative phenylalanine at position 193, a non-native lysine at position196, a non-native threonine at position 199, a non-native aspartic acidat position 203, a non-native glutamic acid or glutamine at position205, a non native aspartic acid at position 208, a non-native glutamicacid or glutamine at position 210, a non native threonine at position214, a non native arginine at position 217 and a non-native cysteine atposition 219, a deletion at position 221, a non-native valine orthreonine at position 222, a deletion at position 223, a non-nativeglutamic acid at position 224, a deletion at position 225, a deletion atposition 235, a deletion at position 221, a non-native valine orthreonine at position 222, a deletion at position 223, a non-nativeglutamic acid at position 224, a deletion at position 225, and adeletion at position 235, a non-native glutamine or glutamic acid atposition 274, a non-native phenylalanine at position 296, a non nativephenylalanine at position 300, a non-native valine at position 309, anon-native glutamic acid at position 320, a non-native glutamic acid atposition 322, a non-native glutamic acid at position 326, a non-nativeglycine at position 327, a non-native glutamic acid at position 334, anon native threonine at position 339, a non native glutamine or glutamicacid at position 355, a non-native serine at position 384, a non-nativeasparagine or glutamic acid at position 392, a non-native methionine atposition 397, a non native glutamic acid at position 419, and a deletionor non-native aspartic acid at position 447.

Taken together, some embodiments utilize variant heavy chain domainswith 0 (when the pI engineering is done in the light constant domainonly), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 22, 23, 26, 27, 28 and 29 mutations (as compared to IgG1) can bemade, as depicted in FIG. 37.

Light Chain pI Variants

In some embodiments, the pI variants are made at least in the lightchain domain of an IgG antibody. In this embodiment, the mutations canbe independently and optionally selected from positions 126, 145, 152,156, 169, 199, 202 and 207. All possible combinations of these 8positions can be made; e.g. a pI antibody may have 1, 2, 3, 4, 5, 6, 7or light constant domain pI mutations. In addition, as is describedherein, any single or combination CL domain mutations can be combinedwith any heavy chain constant domain pI variants.

Specific mutations that find use in lowering the pI of light chainconstant domains include, but are not limited to, a non-native glutamineor glutamic acid at position 126, a non-native glutamine, glutamic acidor threonine at position 145; a non-native aspartic acid at position152, a non-native glutamic acid at position 156, a non-native glutamineor glutamic acid at position 169, a non-native glutamic acid at position199, a non-native glutamic acid at position 202 and a a non-nativeglutamic acid at position 207.

Taken together, some embodiments utilize variant light chain domainswith 0 (when the pI engineering is done in the heavy constant domainonly), 1, 2, 3, 4, 5, 6, or 10 mutations (as compared to Cκ) can bemade, as depicted in FIG. 37.

Heavy and Light Chain pI Variants

As is shown in FIG. 37, a number of pI antibodies have been generatedwith heavy and light chain pI variants. As outlined herein andspecifically meant to be included in the present invention, any pIengineered heavy chain depicted in FIG. 37 and in the sequence listingcan be combined with either a wild-type constant light domain or a pIengineered light constant domain. Similarly, an pI engineered lightchain constant domain can be combined with either a wild-type constantheavy domain or a pI engineered heavy constant domain, even if notspecifically present in FIG. 37. That is, the column of “HC names” and“LC names” are meant to form a matrix, with all possible combinationspossible.

Thus, taken together, any possible combination of the following heavychain constant domain mutations and light chain constant domains can bemade, with each mutation being optionally included or excluded: a) heavychain: a non-native glutamic acid at position 119; a non-native cysteineat position 131; a non-native arginine, lysine or glutamine at position133; a non-native glutamic acid at position 137; a non-native serine atposition 138; a non-native glutamic acid at position 164; a non nativeasparagine at position 192; a non native phenylalanine at position 193,a non-native lysine at position 196, a non-native threonine at position199, a non-native aspartic acid at position 203, a non-native glutamicacid or glutamine at position 205, a non native aspartic acid atposition 208, a non-native glutamic acid or glutamine at position 210, anon native threonine at position 214, a non native arginine at position217 and a non-native cysteine at position 219, a deletion at position221, a non-native valine or threonine at position 222, a deletion atposition 223, a non-native glutamic acid at position 224, a deletion atposition 225, a deletion at position 235, a deletion at position 221, anon-native valine or threonine at position 222, a deletion at position223, a non-native glutamic acid at position 224, a deletion at position225, and a deletion at position 235, a non-native glutamine or glutamicacid at position 274, a non-native phenylalanine at position 296, a nonnative phenylalanine at position 300, a non-native valine at position309, a non-native glutamic acid at position 320, a non-native glutamicacid at position 322, a non-native glutamic acid at position 326, anon-native glycine at position 327, a non-native glutamic acid atposition 334, a non native threonine at position 339, a non nativeglutamine or glutamic acid at position 355, a non-native serine atposition 384, a non-native asparagine or glutamic acid at position 392,a non-native methionine at position 397, a non native glutamic acid atposition 419, and a deletion or non-native aspartic acid at position447; and b) light chain: a non-native glutamine or glutamic acid atposition 126, a non-native glutamine, glutamic acid or threonine atposition 145; a non-native aspartic acid at position 152, a non-nativeglutamic acid at position 156, a non-native glutamine or glutamic acidat position 169, a non-native glutamic acid at position 199, anon-native glutamic acid at position 202 and a a non-native glutamicacid at position 207.

Similarly, the number of mutations that can be generated in suitablepairs of heavy and light constant domains are shown in FIG. 37 (“total #of mutations” column), ranging from 1 to 37.

III. Other Amino Acid Substitutions

As will be appreciated by those in the art, the pI antibodies of theinvention can contain additional amino acid substitutions in addition tothe pI variants.

In some embodiments, amino acid substitutions are imported from oneisotype into the pI antibody despite either a neutrality of charge stateor even an increase of charge state, so as to accommodate the pIvariants. These are sometimes referred to as “non-pI isotypic variants”.For example, the replacement of the native lysine at position 133 ofIgG1 with an arginine from IgG2 is such a change, as is the replacementof the native glutamine in IgG1 at position 196 with the IgG2 lysine,the replacement of native IgG1 proline at position 217 with the IgG2arginine, etc. It should be noted in this instance that as describedabove, pI variants can be made at position 133 as well, substitutingnon-native glutamic acid or glutamine at position 133.

In the hinge region (positions 233-236), changes can be made to increaseeffector function. That is, IgG2 has lowered effector function, and as aresult, amino acid substitutions at these positions from PVA (deletion)can be changed to ELLG, and an additional G327A variant generated aswell.

In the CH3 region, a mutation at position 384 can be made, for examplesubstituting a non-native serine.

Additional mutations that can be made include adding either N-terminalor C-terminal (depending on the structure of the antibody or fusionprotein) “tails” or sequences of one or more low pI amino acids; forexample, glutamic acids and aspartic acids can be added to the CH3C-terminus; generally, from 1 to 5 amino acids are added.

Properties of the pI Antibodies of the Invention

The pI antibodies of the present invention display decreased pIs. Ingeneral, decreases of at least 0.5 log (e.g. corresponding to half a pHpoint) are seen, with decreases of at least about 1, 1.5, 2, 2.5 and 3finding particular use in the invention. The pI can be either calculatedor determined experimentally, as is well known in the art. In addition,it appears that pI antibodies with pIs ranging from 5. to 5.5 to 6exhibit good extended serum half lives. As will be appreciated by thosein the art and depicted in FIG. 30, pIs lower than this are difficult toachieve, as more and more mutations are required and the physical limitsare reached.

The pI antibodies of the present invention display increased serum halflife. As shown in the Figures, surprisingly, every tested pI antibodyhas exhibited an increase in half life as compared to the startingmolecule. While half-life is affected by a number of factors, includingthe Fv portion, increases of 25, 50, 75, 100, 150, 200 and 250% or morecan be obtained using the pI antibodies of the present invention. Asshown in FIG. 34, pI variants can increase half-life from around 4 daysto over 15.

In addition, some variants herein are generated to increase stability.As noted herein, a number of properties of antibodies affect theclearance rate (e.g. stability for half-life) in vivo. In addition toantibody binding to the FcRn receptor, other factors that contribute toclearance and half-life are serum aggregation, enzymatic degradation inthe serum, inherent immunogenicity of the antibody leading to clearingby the immune system, antigen-mediated uptake, FcR (non-FcRn) mediateduptake and non-serum distribution (e.g. in different tissuecompartments).

Accordingly, some additional amino acid substitutions can be made thateffect one or more of these properties. As shown in FIG. 37, thisinclude, but are not limited to, 222K, 274K, 296Y, 300Y, 339A, 355R,384N, 392K, 397V, 419Q, 296Y/300Y, 384N/392K/397V, 137G, 138G, 192S,193L, 1991, 203N, 214K, 137G/138G, 192S/193G, 1991/203N, 214K/222K,138G/192S/193L and 137G/138G/192S/193L.

IV. Optional and Additional Fc Engineering

FcRn Modifications

In some embodiments, the pI variants of the present invention can becombined with amino acid substitutions in the FcRn binding domain.Surprisingly, the present invention shows that pI variants can beindependently and optionally combined with Fc variants that result inboth higher binding to the FcRn receptor as well as increasedhalf-lives.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless other wise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. In some cases,the FcRn variants bind to the human FcRn receptor, or it may bedesirable to design variants that bind to rodent or primate receptors inaddition, to facilitate clinical trials.

A variety of such substitutions are known and described in U.S. Ser. No.12/341,769. In some embodiments, a pI antibody can be engineered toinclude any of the following substitutions, alone or in any combination:436I, 436V, 311I, 311V, 428L, 434S, 428L/434S, 259I, 308F, 259I/308F,259I/308F/428L, 307Q/434S, 434A, 434H, 250Q/428L, M252Y/S254T/T256E,307Q/434A, 307Q//380A/434A, and 308P/434A. Numbering is EU as in Kabat,and it is understood that the substitution is non-native to the startingmolecule. As has been shown previously, these FcRn substitutions work inIgG1, IgG2 and IgG1/G2 hybrid backbones, and are specifically includedfor IgG3 and IgG4 backbones and derivatives of any IgG isoform as well.

In some embodiments, it is also possible to do pI engineering onvariable regions, either framework or CDRs, as is generally described inUS Publication 2011/0076275, expressly incorporated herein by reference.

In other embodiments, no pI variants are made in the variable region(s)of the antibodies, e.g. no amino acid substitutions are made thatpurposefully decrease the pI of the amino acid at a position, nor of thetotal protein. This is to be distinguished from affinity maturationsubstitutions in the variable region(s) that are made to increasebinding affinity of the antibody to its antigen but may result in alower pI amino acid being added. That is, a pI variant in the variableregion(s) is generally significantly “silent” with respect to bindingaffinity.

Fc Engineering

In addition to substitutions made to increase binding affinity to FcRnand/or increase serum half life, other substitutions can be made in theFc region, in general for altering binding to FcγR receptors.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65,entirely incorporated by reference), as well as any undiscovered humanFcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism,including but not limited to humans, mice, rats, rabbits, and monkeys.Mouse FcγRs include but are not limited to FcγRI (CD64), FcγRII (CD32).FcγRIII-1 (CD16), and FcγRIII-2 (CD16-2), as well as any undiscoveredmouse FcγRs or FcγR isoforms or allotypes.

There are a number of useful Fc substitutions that can be made to alterbinding to one or more of the FcγR receptors. Substitutions that resultin increased binding as well as decreased binding can be useful. Forexample, it is known that increased binding to FcγRIIIa generallyresults in increased ADCC (antibody dependent cell-mediatedcytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. Similarly, decreasedbinding to FcγRIIb (an inhibitory receptor) can be beneficial as well insome circumstances. Amino acid substitutions that find use in thepresent invention include those listed in U.S. Ser. No. 11/124,620(particularly FIG. 41), Ser. Nos. 11/174,287, 11/396,495, 11/538,406,all of which are expressly incorporated herein by reference in theirentirety and specifically for the variants disclosed therein. Particularvariants that find use include, but are not limited to, 236A, 239D,239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E,239D/332E/330Y, 239D, 332E/330L and 299T.

V. Other Antibody Modifications

Affinity Maturation

In some embodiments, one or more amino acid modifications are made inone or more of the CDRs of the antibody. In general, only 1 or 2 or 3amino acids are substituted in any single CDR, and generally no morethan from 4, 5, 6, 7, 8 9 or 10 changes are made within a set of CDRs.However, it should be appreciated that any combination of nosubstitutions, 1, 2 or 3 substitutions in any CDR can be independentlyand optionally combined with any other substitution.

In some cases, amino acid modifications in the CDRs are referred to as“affinity maturation”. An “affinity matured” antibody is one having oneor more alteration(s) in one or more CDRs which results in animprovement in the affinity of the antibody for antigen, compared to aparent antibody which does not possess those alteration(s). In somecases, although rare, it may be desirable to decrease the affinity of anantibody to its antigen, but this is generally not preferred.

Affinity maturation can be done to increase the binding affinity of theantibody for the antigen by at least about 10% to 50-100-150% or more,or from 1 to 5 fold as compared to the “parent” antibody. Preferredaffinity matured antibodies will have nanomolar or even picomolaraffinities for the target antigen. Affinity matured antibodies areproduced by known procedures. See, for example, Marks et al., 1992.Biotechnology 10:779-783 that describes affinity maturation by variableheavy chain (VH) and variable light chain (VL) domain shuffling. Randommutagenesis of CDR and/or framework residues is described in: Barbas, etal. 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995.Gene 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004;Jackson et al., 1995. J. Immunol. 154(7):3310-9; and Hawkins et al,1992, J. Mol. Biol. 226:889-896, for example.

Alternatively, amino acid modifications can be made in one or more ofthe CDRs of the antibodies of the invention that are “silent”, e.g. thatdo not significantly alter the affinity of the antibody for the antigen.These can be made for a number of reasons, including optimizingexpression (as can be done for the nucleic acids encoding the antibodiesof the invention).

Thus, included within the definition of the CDRs and antibodies of theinvention are variant CDRs and antibodies; that is, the antibodies ofthe invention can include amino acid modifications in one or more of theCDRs of Ab79 and Ab19. In addition, as outlined below, amino acidmodifications can also independently and optionally be made in anyregion outside the CDRs, including framework and constant regions.

ADC Modifications

In some embodiments, the pI antibodies of the invention are conjugatedwith drugs to form antibody-drug conjugates (ADCs). In general, ADCs areused in oncology applications, where the use of antibody-drug conjugatesfor the local delivery of cytotoxic or cytostatic agents allows for thetargeted delivery of the drug moiety to tumors, which can allow higherefficacy, lower toxicity, etc. An overview of this technology isprovided in Ducry et al., Bioconjugate Chem., 21:5-13 (2010). Carter etal., Cancer J. 14(3):154 (2008) and Senter, Current Opin. Chem. Biol.13:235-244 (2009), all of which are hereby incorporated by reference intheir entirety

Thus the invention provides pI antibodies conjugated to drugs.Generally, conjugation is done by covalent attachment to the antibody,as further described below, and generally relies on a linker, often apeptide linkage (which, as described below, may be designed to besensitive to cleavage by proteases at the target site or not). Inaddition, as described above, linkage of the linker-drug unit (LU-D) canbe done by attachment to cysteines within the antibody. As will beappreciated by those in the art, the number of drug moieties perantibody can change, depending on the conditions of the reaction, andcan vary from 1:1 to 10:1 drug:antibody. As will be appreciated by thosein the art, the actual number is an average.

Thus the invention provides pI antibodies conjugated to drugs. Asdescribed below, the drug of the ADC can be any number of agents,including but not limited to cytotoxic agents such as chemotherapeuticagents, growth inhibitory agents, toxins (for example, an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (that is, a radioconjugate) areprovided. In other embodiments, the invention further provides methodsof using the ADCs.

Drugs for use in the present invention include cytotoxic drugs,particularly those which are used for cancer therapy. Such drugsinclude, in general DNA damaging agents, anti-metabolites, naturalproducts and their analogs. Exemplary classes of cytotoxic agentsinclude the enzyme inhibitors such as dihydrofolate reductaseinhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNAcleavers, topoisomerase inhibitors, the anthracycline family of drugs,the vinca drugs, the mitomycins, the bleomycins, the cytotoxicnucleosides, the pteridine family of drugs, diynenes, thepodophyllotoxins, dolastatins, maytansinoids, differentiation inducers,and taxols.

Members of these classes include, for example, methotrexate,methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin,daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin,aminopterin, tallysomycin, podophyllotoxin and podophyllotoxinderivatives such as etoposide or etoposide phosphate, vinblastine,vincristine, vindesine, taxanes including taxol, taxotere retinoic acid,butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin,esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,camptothecin, maytansinoids (including DMI), monomethylauristatin E(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and theiranalogues.

Toxins may be used as antibody-toxin conjugates and include bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).Toxins may exert their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of an pI antibody and one or more small molecule toxins, suchas a maytansinoids, dolastatins, auristatins, a trichothecene,calicheamicin, and CC1065, and the derivatives of these toxins that havetoxin activity, are contemplated.

Maytansinoids

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol andmaytansinol analogues prepared synthetically according to known methods.As described below, drugs may be modified by the incorporation of afunctionally active group such as a thiol or amine group for conjugationto the antibody.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/−dechloro (U.S. Pat. No. 4,294,757) (prepared byacylation using acyl chlorides) and those having modifications at otherpositions

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H2S or P2S5);C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Of particular use are DM1 (disclosed in U.S. Pat. No. 5,208,020,incorporated by reference) and DM4 (disclosed in U.S. Pat. No.7,276,497, incorporated by reference). See also a number of additionalmaytansinoid derivatives and methods in U.S. Pat. No. 5,416,064,WO/01/24763, U.S. Pat. Nos. 7,303,749, 7,601,354, U.S. Ser. No.12/631,508, WO02/098883, U.S. Pat. Nos. 6,441,163, 7,368,565, WO02/16368and WO04/1033272, all of which are expressly incorporated by referencein their entirety.

ADCs containing maytansinoids, methods of making same, and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020;5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCscomprising a maytansinoid designated DM1 linked to the monoclonalantibody C242 directed against human colorectal cancer. The conjugatewas found to be highly cytotoxic towards cultured colon cancer cells,and showed antitumor activity in an in vivo tumor growth assay.

Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×105 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Auristatins and Dolastatins

In some embodiments, the ADC comprises an pI antibody conjugated todolastatins or dolostatin peptidic analogs and derivatives, theauristatins (U.S. Pat. Nos. 5,635,483; 5,780,588). Dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin orauristatin drug moiety may be attached to the antibody through the N(amino) terminus or the C (carboxyl) terminus of the peptidic drugmoiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in “Senter etal, Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented Mar. 28, 2004 and described in UnitedStates Patent Publication No. 2005/0238648, the disclosure of which isexpressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE (shown in FIG. 10 wherein thewavy line indicates the covalent attachment to a linker (L) of anantibody drug conjugate; see U.S. Pat. No. 6,884,869 expresslyincorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF, shown in FIG. 10wherein the wavy line indicates the covalent attachment to a linker (L)of an antibody drug conjugate (US 2005/0238649, U.S. Pat. Nos. 5,767,237and 6,124,431, expressly incorporated by reference in their entirety):

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 8):

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lubke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol21(7):778-784.

Calicheamicin

In other embodiments, the ADC comprises an antibody of the inventionconjugated to one or more calicheamicin molecules. For example, Mylotargis the first commercial ADC drug and utilizes calicheamicin γ1 as thepayload (see U.S. Pat. No. 4,970,198, incorporated by reference in itsentirety). Additional calicheamicin derivatives are described in U.S.Pat. Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001,5,767,285 and 5,877,296, all expressly incorporated by reference. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ1I, α2I, α2I, N-acetyl-γ1I, PSAG and θ11 (Hinman et al.,Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Duocarmycins

CC-1065 (see U.S. Pat. No. 4,169,888, incorporated by reference) andduocarmycins are members of a family of antitumor antibiotics utilizedin ADCs. These antibiotics appear to work through sequence-selectivelyalkylating DNA at the N3 of adenine in the minor groove, which initiatesa cascade of events that result in apoptosis.

Important members of the duocarmycins include duocarmycin A (U.S. Pat.No. 4,923,990, incorporated by reference) and duocarmycin SA (U.S. Pat.No. 5,101,038, incorporated by reference), and a large number ofanalogues as described in U.S. Pat. Nos. 7,517,903, 7,691,962,5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,070,092; 5,641,780;5,101,038; 5,084,468, 5,475,092, 5,585,499, 5,846,545, WO2007/089149,WO2009/017394A1, U.S. Pat. Nos. 5,703,080, 6,989,452, 7,087,600,7,129,261, 7,498,302, and 7,507,420, all of which are expresslyincorporated by reference.

VI. Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an ADC formed between anantibody and a compound with nucleolytic activity (e.g., a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt211, I1131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lu.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc99m or I123, Re186, Re188 and In111 can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate Iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

For compositions comprising a plurality of antibodies, the drug loadingis represented by p, the average number of drug molecules per Antibody.Drug loading may range from 1 to 20 drugs (D) per Antibody. The averagenumber of drugs per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution ofAntibody-Drug-Conjugates in terms of p may also be determined.

In some instances, separation, purification, and characterization ofhomogeneous Antibody-Drug-conjugates where p is a certain value fromAntibody-Drug-Conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis. In exemplaryembodiments, p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof.

The generation of Antibody-drug conjugate compounds can be accomplishedby any technique known to the skilled artisan. Briefly, theAntibody-drug conjugate compounds can include an pI antibody as theAntibody unit, a drug, and optionally a linker that joins the drug andthe binding agent.

A number of different reactions are available for covalent attachment ofdrugs and/or linkers to binding agents. This is can be accomplished byreaction of the amino acid residues of the binding agent, for example,antibody molecule, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.A commonly used non-specific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of an antibody molecule.

Also available for attachment of drugs to binding agents is the Schiffbase reaction. This method involves the periodate oxidation of a drugthat contains glycol or hydroxy groups, thus forming an aldehyde whichis then reacted with the binding agent. Attachment occurs via formationof a Schiff base with amino groups of the binding agent. Isothiocyanatescan also be used as coupling agents for covalently attaching drugs tobinding agents. Other techniques are known to the skilled artisan andwithin the scope of the present invention.

In some embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. In otherembodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with anpI antibody of the invention under appropriate conditions.

It will be understood that chemical modifications may also be made tothe desired compound in order to make reactions of that compound moreconvenient for purposes of preparing conjugates of the invention. Forexample a functional group e.g. amine, hydroxyl, or sulfhydryl, may beappended to the drug at a position which has minimal or an acceptableeffect on the activity or other properties of the drug

VII. Linker Units

Typically, the antibody-drug conjugate compounds comprise a Linker unitbetween the drug unit and the antibody unit. In some embodiments, thelinker is cleavable under intracellular or extracellular conditions,such that cleavage of the linker releases the drug unit from theantibody in the appropriate environment. For example, solid tumors thatsecrete certain proteases may serve as the target of the cleavablelinker, in other embodiments, it is the intracellular proteases that areutilized. In yet other embodiments, the linker unit is not cleavable andthe drug is released, for example, by antibody degradation in lysosomes.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (for example, within a lysosomeor endosome or caveolea). The linker can be, for example, a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease. Insome embodiments, the peptidyl linker is at least two amino acids longor at least three amino acids long or more.

Cleaving agents can include, without limitation, cathepsins B and D andplasmin, all of which are known to hydrolyze dipeptide drug derivativesresulting in the release of active drug inside target cells (see, e.g.,Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyllinkers that are cleavable by enzymes that are present inCD38-expressing cells. For example, a peptidyl linker that is cleavableby the thiol-dependent protease cathepsin-B, which is highly expressedin cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Glylinker (SEQ ID NO: X)). Other examples of such linkers are described,e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference inits entirety and for all purposes.

In some embodiments, the peptidyl linker cleavable by an intracellularprotease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat.No. 6,214,345, which describes the synthesis of doxorubicin with theval-cit linker).

In other embodiments, the cleavable linker is pH-sensitive, that is,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (for example,a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) may be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989. Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (for example, a disulfide linker). A variety of disulfidelinkers are known in the art, including, for example, those that can beformed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-2-pyridyl-dithio)toluene)-,SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No.2005/0238649 incorporated by reference herein in its entirety and forall purposes).

In many embodiments, the linker is self-immolative. As used herein, theterm “self-immolative Spacer” refers to a bifunctional chemical moietythat is capable of covalently linking together two spaced chemicalmoieties into a stable tripartite molecule. It will spontaneouslyseparate from the second chemical moiety if its bond to the first moietyis cleaved. See for example, WO 2007059404A2, WO06110476A2,WO05112919A2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431,WO04/043493 and WO02/083180, which are directed to drug-cleavablesubstrate conjugates where the drug and cleavable substrate areoptionally linked through a self-immolative linker and which are allexpressly incorporated by reference.

Often the linker is not substantially sensitive to the extracellularenvironment. As used herein, “not substantially sensitive to theextracellular environment.” in the context of a linker, means that nomore than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of thelinkers, in a sample of antibody-drug conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (for example, in plasma).

Whether a linker is not substantially sensitive to the extracellularenvironment can be determined, for example, by incubating with plasmathe antibody-drug conjugate compound for a predetermined time period(for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amountof free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (thatis, in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the auristatin compound and the pI antibodies of theinvention.

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO 2004-010957. U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No. 2006/0024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

VIII. Drug Loading

Drug loading is represented by p and is the average number of Drugmoieties per antibody in a molecule. Drug loading (“p”) may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moremoieties (D) per antibody, although frequently the average number is afraction or a decimal. Generally, drug loading of from 1 to 4 isfrequently useful, and from 1 to 2 is also useful. ADCs of the inventioninclude collections of antibodies conjugated with a range of drugmoieties, from 1 to 20. The average number of drug moieties per antibodyin preparations of ADC from conjugation reactions may be characterizedby conventional means such as mass spectroscopy and, ELISA assay.

The quantitative distribution of ADC in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADC where p is a certain value from ADCwith other drug loadings may be achieved by means such aselectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See US 2005-0238649A1 (herein incorporated by reference in its entirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachments (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography.

In some embodiments, a homogeneous ADC with a single loading value maybe isolated from the conjugation mixture by electrophoresis orchromatography.

Methods of Determining Cytotoxic Effect of ADCs

Methods of determining whether a Drug or Antibody-Drug conjugate exertsa cytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of an Antibody Drug conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theAntibody Drug conjugate in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Antibody Drug conjugate.

For determining whether an Antibody Drug conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of ³H-thymidine into cells of the culture is measured inthe presence and absence of the Antibody Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that an Antibody Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytoxicity (Skehan et al., 1990, J. Natl.Cancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983. J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., 1995, CancerResearch 55:3110-16).

In vivo, the effect of a therapeutic composition of the pI antibody ofthe invention can be evaluated in a suitable animal model. For example,xenogenic cancer models can be used, wherein cancer explants or passagedxenograft tissues are introduced into immune compromised animals, suchas nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408).Efficacy can be measured using assays that measure inhibition of tumorformation, tumor regression or metastasis, and the like.

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally.Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980).

Glycosylation

Another type of covalent modification is alterations in glycosylation.In another embodiment, the antibodies disclosed herein can be modifiedto include one or more engineered glycoforms. By “engineered glycoform”as used herein is meant a carbohydrate composition that is covalentlyattached to the antibody, wherein said carbohydrate composition differschemically from that of a parent antibody. Engineered glycoforms may beuseful for a variety of purposes, including but not limited to enhancingor reducing effector function. A preferred form of engineered glycoformis afucosylation, which has been shown to be correlated to an increasein ADCC function, presumably through tighter binding to the FcγRIIIareceptor. In this context, “afucosylation” means that the majority ofthe antibody produced in the host cells is substantially devoid offucose, e.g. 90-95-98% of the generated antibodies do not haveappreciable fucose as a component of the carbohydrate moiety of theantibody (generally attached at N297 in the Fc region). Definedfunctionally, afucosylated antibodies generally exhibit at least a 50%or higher affinity to the FcγRIIIa receptor.

Engineered glycoforms may be generated by a variety of methods known inthe art (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO02/30954A1, all entirely incorporated by reference; (Potelligent®technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylationengineering technology [Glycart Biotechnology AG, Zuirich,Switzerland]). Many of these techniques are based on controlling thelevel of fucosylated and/or bisecting oligosaccharides that arecovalently attached to the Fc region, for example by expressing an IgGin various organisms or cell lines, engineered or otherwise (for exampleLee-13 CHO cells or rat hybridoma YB2/0 cells, by regulating enzymesinvolved in the glycosylation pathway (for example FUT8[α1,6-fucosyltranserase] and/or β1-4-N-acetylglucosaminyltransferase III[GnTIII]), or by modifying carbohydrate(s) after the IgG has beenexpressed. For example, the “sugar engineered antibody” or “SEAtechnology” of Seattle Genetics functions by adding modified saccharidesthat inhibit fucosylation during production; see for example20090317869, hereby incorporated by reference in its entirety.Engineered glycoform typically refers to the different carbohydrate oroligosaccharide; thus an antibody can include an engineered glycoform.

Alternatively, engineered glycoform may refer to the IgG variant thatcomprises the different carbohydrate or oligosaccharide. As is known inthe art, glycosylation patterns can depend on both the sequence of theprotein (e.g., the presence or absence of particular glycosylation aminoacid residues, discussed below), or the host cell or organism in whichthe protein is produced. Particular expression systems are discussedbelow.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thestarting sequence (for O-linked glycosylation sites). For ease, theantibody amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both entirelyincorporated by reference.

Removal of carbohydrate moieties present on the starting antibody (e.g.post-translationally) may be accomplished chemically or enzymatically.Chemical deglycosylation requires exposure of the protein to thecompound trifluoromethanesulfonic acid, or an equivalent compound. Thistreatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the polypeptide intact. Chemical deglycosylation is described byHakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge etal., 1981, Anal. Biochem. 118:131, both entirely incorporated byreference. Enzymatic cleavage of carbohydrate moieties on polypeptidescan be achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., 1987, Meth. Enzymol. 138:350, entirelyincorporated by reference. Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al., 1982. J. Biol. Chem. 257:3105, entirelyincorporated by reference. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in, for example,2005-2006 PEG Catalog from Nektar Therapeutics (available at the Nektarwebsite) U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337, all entirely incorporated by reference. Inaddition, as is known in the art, amino acid substitutions may be madein various positions within the antibody to facilitate the addition ofpolymers such as PEG. See for example, U.S. Publication No.2005/0114037A1, entirely incorporated by reference.

Nucleic Acids and Host Cells

Included within the invention are the nucleic acids encoding the pIantibodies of the invention. In the case where both a heavy and lightchain constant domains are included in the pI antibody, generally theseare made using nucleic acids encoding each, that are combined intostandard host cells (e.g. CHO cells, etc.) to produce the tetramericstructure of the antibody. If only one pI engineered constant domain isbeing made, only a single nucleic acid will be used.

IX Antibody Compositions for in Vivo Administration

The use of the pI antibodies of the invention in therapy will depend onthe antigen binding component; e.g. in the case of full length standardtherapeutic antibodies, on the antigen to which the antibody's Fv binds.That is, as will be appreciated by those in the art, the treatment ofspecific diseases can be done with the additional benefit of increasedhalf life of the molecule. This can result in a variety of benefits,including, but not limited to, less frequent dosing (which can lead tobetter patient compliance), lower dosing, and lower production costs.

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to provide antibodies with otherspecificities. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine, growth inhibitory agent and/orsmall molecule antagonist. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration should besterile, or nearly so. This is readily accomplished by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gamma.ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

When encapsulated antibodies remain in the body for a long time, theymay denature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

X. Administrative Modalities

The antibodies and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

XI. Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Thus for B cell tumors, the subject may experience a decrease in theso-called B symptoms, i.e., night sweats, fever, weight loss, and/orurticaria. For pre-malignant conditions, therapy with an pI therapeuticagent may block and/or prolong the time before development of a relatedmalignant condition, for example, development of multiple myeloma insubjects suffering from monoclonal gammopathy of undeterminedsignificance (MGUS).

An improvement in the disease may be characterized as a completeresponse. By “complete response” is intended an absence of clinicallydetectable disease with normalization of any previously abnormalradiographic studies, bone marrow, and cerebrospinal fluid (CSF) orabnormal monoclonal protein in the case of myeloma.

Such a response may persist for at least 4 to 8 weeks, or sometimes 6 to8 weeks, following treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number ofmalignant cells present in the subject, or the measured bulk of tumormasses or the quantity of abnormal monoclonal protein) in the absence ofnew lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the pI antibodies usedin the present invention depend on the disease or condition to betreated and may be determined by the persons skilled in the art.

An exemplary, non-limiting range for a therapeutically effective amountof an pI antibody used in the present invention is about 0.1-100 mg/kg,such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such asabout 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1,or about 3 mg/kg. In another embodiment, the antibody is administered ina dose of 1 mg/kg or more, such as a dose of from 1 to 20 mg/kg, e.g. adose of from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.

A medical professional having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, a physician or a veterinarian couldstart doses of the medicament employed in the pharmaceutical compositionat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved.

In one embodiment, the pI antibody is administered by infusion in aweekly dosage of from 10 to 500 mg/kg such as of from 200 to 400 mg/kgSuch administration may be repeated, e.g., 1 to 8 times, such as 3 to 5times. The administration may be performed by continuous infusion over aperiod of from 2 to 24 hours, such as of from 2 to 12 hours.

In one embodiment, the pI antibody is administered by slow continuousinfusion over a long period, such as more than 24 hours, if required toreduce side effects including toxicity.

In one embodiment the pI antibody is administered in a weekly dosage offrom 250 mg to 2000 mg, such as for example 300 mg, 500 mg, 700 mg, 1000mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4 to 6 times.The administration may be performed by continuous infusion over a periodof from 2 to 24 hours, such as of from 2 to 12 hours. Such regimen maybe repeated one or more times as necessary, for example, after 6 monthsor 12 months. The dosage may be determined or adjusted by measuring theamount of compound of the present invention in the blood uponadministration by for instance taking out a biological sample and usinganti-idiotypic antibodies which target the antigen binding region of thepI antibody.

In a further embodiment, the pI antibody is administered once weekly for2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8 weeks.

In one embodiment, the pI antibody is administered by maintenancetherapy, such as, e.g., once a week for a period of 6 months or more.

In one embodiment, the pI antibody is administered by a regimenincluding one infusion of an pI antibody followed by an infusion of anpI antibody conjugated to a radioisotope. The regimen may be repeated,e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of an antibody in an amount of about0.1-100 mg % kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on atleast one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

In some embodiments the pI antibody molecule thereof is used incombination with one or more additional therapeutic agents, e.g. achemotherapeutic agent. Non-limiting examples of DNA damagingchemotherapeutic agents include topoisomerase I inhibitors (e.g.,irinotecan, topotecan, camptothecin and analogs or metabolites thereof,and doxorubicin); topoisomerase II inhibitors (e.g., etoposide,teniposide, and daunorubicin); alkylating agents (e.g., melphalan,chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine,semustine, streptozocin, decarbazine, methotrexate, mitomycin C, andcyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, andcarboplatin); DNA intercalators and free radical generators such asbleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitibine,gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine,pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include:paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, andrelated analogs; thalidomide, lenalidomide, and related analogs (e.g.,CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinibmesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κBinhibitors, including inhibitors of IκB kinase; antibodies which bind toproteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,over-expressed or activated in cancers, the inhibition of whichdownregulates cell replication.

In some embodiments, the antibodies of the invention can be used priorto, concurrent with, or after treatment with Velcade® (bortezomib).

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

Example 1 Design of Non-Native Charge Substitutions to Reduce pI

Antibody constant chains were modified with lower pI by engineeringsubstitutions in the constant domains. Reduced pI can be engineered bymaking substitutions of basic amino acids (K or R) to acidic amino acids(D or E), which result in the largest decrease in pI. Mutations of basicamino acids to neutral amino acids and neutral amino acids to acidicamino acids will also result in a decrease in pI. A list of amino acidpK values can be found in Table 1 of Bjellqvist et al., 1994,Electrophoresis 15:529-539.

We chose to explore substitutions in the antibody CH1 (Cγ1) and CL(Ckappa or CK) regions (sequences are shown in FIG. 1) because, unlikethe Fc region, they do not interact with native ligands that impact theantibody's pharmacological properties. In deciding which positions tomutate, the surrounding environment and number of contacts the WT aminoacid makes with its neighbors was taken into account such as to minimizethe impact of a substitution or set of substitutions on structure and/orfunction. The solvent accessibility or fraction exposed of each CH1 andCK position was calculated using relevant crystal structures of antibodyFab domains. The results are shown in FIGS. 2 and 3 for the Cγ1 and CKrespectively. Design was guided further by examining the CH1 and CLdomains for positions that are isotypic between the immunoglobulinisotypes (IgG1, IgG2, IgG3, and IgG4). Because such variations occurnaturally, such position are expected to be amenable to substitution.Based on this analysis, a number of substitutions were identified thatreduce pI but are predicted to have minimal impact on the biophysicalproperties of the domains.

Example 2 Anti-VEGF Antibodies with Engineered CH1 and CK Regions HavingLower pI

Amino acid modifications were engineered in the CH1 and CK domains of anIgG1 antibody to lower the pI of the antibody. Based on the aboveanalysis, chosen substitutions for the heavy chain CH1 were 119E, 133E,164E, 205E, 208D, and 210E, and substitutions for the light chain Cκsubstitutions were 126E, 145E, 152D, 156E, 169E, and 202E. These variantconstant chains are referred to as IgG1-CH1-pI(6) and CK-pI(6)respectively, and their amino acid sequences are provided in FIG. 4.

CH1 and CK variants were engineered in the context of an antibodytargeting vascular endothelial factor (VEGF). The heavy and light chainvariable regions (VH and VL) are those of a humanized version of theantibody A4.6.1, also referred to as bevacizumab (Avastin®), which isapproved for the treatment of a variety of cancers. These variableregion sequences are provided in FIG. 5. The anti-VEGF antibody variantcontaining the low pI substitutions is referred to as XENP9493Bevacizumab-IgG1-CH1-pI(6)-CK-pI(6), and the amino acid sequences of theheavy and light chains of this antibody are provided in FIG. 6. Astructural model of the Fab domain showing the 6 substitutions ofCH1-pI(6) and the 6 substitutions of CK-pI(6) is shown in FIG. 7. Thecalculated pI of WT anti-VEGF (bevacizumab) is 8.14. The calculated pIof the engineered anti-VEGF CH1 variant is 6.33 and that of theanti-VEGF CK variant is 6.22. When the heavy chain and light chain pIengineered anti-VEGF variants are co-transfected, the full-lengthanti-VEGF mAb has a calculated pI of 5.51.

Genes encoding the heavy and light chains of the anti-VEGF antibodieswere constructed in the mammalian expression vector pTT5. The human IgG1constant chain gene was obtained from IMAGE clones and subcloned intothe pTT5 vector. VH and VL genes encoding the anti-VEGF antibodies weresynthesized commercially (Blue Heron Biotechnologies, Bothell Wash.),and subcloned into the vectors encoding the appropriate CL and IgG1constant chains. Amino acid modifications were constructed usingsite-directed mutagenesis using the QuikChange® site-directedmutagenesis methods (Stratagene, La Jolla Calif.). All DNA was sequencedto confirm the fidelity of the sequences.

Plasmids containing heavy chain gene (VH-Cγ1-Cγ2-Cγ3) wereco-transfected with plasmid containing light chain gene (VL-Cκ) into293E cells using Ilipofectamine (Invitrogen, Carlsbad Calif.) and grownin FreeStyle 293 media (Invitrogen, Carlsbad Calif.). After 5 days ofgrowth, the antibodies were purified from the culture supernatant byprotein A affinity using the MabSelect resin (GE Healthcare). Antibodyconcentrations were determined by bicinchoninic acid (BCA) assay(Pierce).

The pI engineered anti-VEGF mAbs were characterized by SDS PAGE on anAgilent Bioanalyzer (FIG. 8), by size exclusion chromatography (SEC)(FIG. 9), isoelectric focusing (IEF) gel electrophoresis (FIG. 10),binding to antigen VEGF by Biacore (FIG. 11), and differential scanningcalorimetry (DSC) (FIG. 12). All mAbs showed high purity on SDS-PAGE andSEC. IEF gels indicated that each variant had the designed isoelectricpoint. VEGF binding analysis on Biacore showed that pI engineeredanti-VEGF bound to VEGF with similar affinity as bevacizumab, indicatingthat the designed substitutions did not perturb the function of the mAb.DSC showed that the anti-VEGF variant with both CH1 and CL engineeredsubstitutions had high thermostability with a Tm of 71.9° C.

Pharmacokinetic experiments were performed in B6 mice that arehomozygous knock-outs for murine FcRn and heterozygous knock-ins ofhuman FcRn (mFcRn^(−/−), hFcRn⁺) (Petkova et al., 2006, Int Immunol18(12):1759-69, entirely incorporated by reference), herein referred toas hFcRn or hFcRn⁺ mice. Samples tested included the parent IgG1/2constant region, the pI-engineered variant with a pI of 5.51, referredto as IgG1_CH-CL_pI_eng, and an Fc variant version of IgG1/2 containingthe substitution N434S, which improves affinity to human FcRn.

A single, intravenous tail vein injection of anti-VEGF antibody (2mg/kg) was given to groups of 4-7 female mice randomized by body weight(20-30 g range). Blood (˜50 ul) was drawn from the orbital plexus ateach time point, processed to serum, and stored at −80° C. untilanalysis. Antibody concentrations were determined using an ELISA assay.Serum concentration of antibody was measured using a recombinant VEGF(VEGF-165, PeproTech, Rocky Hill, N.J.) as capture reagent, anddetection was carried out with biotinylated anti-human kappa antibodyand europium-labeled streptavidin. The time resolved fluorescence signalwas collected. PK parameters were determined for individual mice with anon-compartmental model using WinNonLin (Pharsight Inc. Mountain ViewCalif.). Nominal times and dose were used with uniform weighing ofpoints.

Results are shown in FIG. 13. Fitted half-life (t½) values, whichrepresents the beta phase that characterizes elimination of antibodyfrom serum, are shown in Table 1. The pI-engineered variant, containingsubstitutions in CH1 and CL that reduce the pI, extended half-life to7.4 days, an improvement of approximately 2.6-fold relative to IgG1/2.The pI-engineered variant had a comparable half-life to the Fc variantversion N434S. Combinations of antibody variants are contemplated thatreduce pI and improve affinity for FcRn for extending the half-lives ofantibodies and Fc fusions.

TABLE 1 PK results of pI-engineered variant Individual mice Average t1/2(days) t1/2 St. Dev. Group Variant n n1 n2 n3 n4 (days) (days) 7349IgG1/2_WT 4 2.9 2.5 3.2 2.8 2.9 0.3 7350 IgG1/2_N434S 4 6.3 7.7 7.3 6.57.0 0.7 9493 IgG1_CH-CL_pI_eng 3 7.4 8.4 6.4 7.4 1.0

Example 3 PK Analysis of IgG Constant Regions

PK studies of IgG1 and IgG2 isotype versions of bevacizumab were carriedout in the huFcRn mice as described above. The IgG1 results from fourseparate PK studies are shown in FIG. 14. The half-lives from the fourstudies were 3.0, 3.9, 2.8, and 2.9 days, resulting in an averagehalf-life of 3.2 days. The PK results from the IgG2 study are shown inFIG. 15. The half-life of IgG2 was 5.9 days.

The PK results from the IgG1 and IgG2 were analyzed with the resultsfrom the IgG1/2 and pI-engineered versions of bevacizumab. Table 2 showsthe half-lives of the antibodies along with their calculated pI. Thesedata are plotted in FIG. 16.

TABLE 2 PK results of antibodies with identical Fv (bevacizumab) butconstant regions with different pI's Average XENP IgG pI t 1/2 (days)4547 IgG1 8.1 3.2 7349 IgG1/2 8.1 2.9 6384 IgG2 7.3 5.9 9493IgG1_CH-CL_pI_eng 5.6 7.4 [aka IgG1-pI (12)]

A correlation was observed between half-life and the pI of theantibodies. These data further suggest that engineering of antibodyconstant chains, including heavy and light chain constant regions, forreduced isoelectric point is potentially a novel generalizable approachto extending the serum half-lives of antibodies and Fc fusions.

Example 4 Engineering Approaches to Constant Region pI Engineering

Reduction in the pI of a protein or antibody can be carried out using avariety of approaches. At the most basic level, residues with high pKa's(lysine, arginine, and to some extent histidine) are replaced withneutral or negative residues, and/or neutral residues are replaced withlow pKa residues (aspartic acid and glutamic acid). The particularreplacements may depend on a variety of factors, including location inthe structure, role in function, and immunogenicity.

Because immunogenicity is a concern, efforts can be made to minimize therisk that a substitution that lowers the pI will elicit immunogenicity.One way to minimize risk is to minimize the mutational load of thevariants, i.e. to reduce the pI with the fewest number of mutations.Charge swapping mutations, where a K. R, or H is replaced with a D or E,have the greatest impact on reducing pI, and so these substitutions arepreferred. Another approach to minimizing the risk of immunogenicitywhile reducing pI is to utilize substitutions from homologous humanproteins. Thus for antibody constant chains, the isotypic differencesbetween the IgG subclasses (IgG1, IgG2, IgG3, and IgG4) provide low-risksubstitutions. Because immune recognition occurs at a local sequencelevel, i.e. MHC II and T-cell receptors recognize epitopes typically 9residues in length, pI-altering substitutions may be accompanied byisotypic substitutions proximal in sequence. In this way, epitopes canbe extended to match a natural isotype. Such substitutions would thusmake up epitopes that are present in other human IgG isotypes, and thuswould be expected to be tolerized.

FIG. 17 shows an amino acid sequence alignment of the IgG subclasses.Residues with a bounded box illustrate isotypic differences between theIgG's. Residues which contribute to a higher pI (K, R, and H) or lowerpI (D and E) are highlighted in bold. Designed substitutions that eitherlower the pI, or extend an epitope to match a natural isotype are shownin gray.

FIG. 18 shows the amino acid sequence of the CK and Cλ light constantchains. Homology between Cκ and Cλ is not as high as between the IgGsubclasses. Nonetheless the alignment may be used to guidesubstitutions. Residues which contribute to a higher pI (K, R, and H) orlower pI (D and E) are highlighted in bold. Gray indicates lysine,arginines, and histidines that may be substituted, preferably withaspartic or glutatmic acids, to lower the isoelectric point.

Another approach to engineering lower pI into proteins and antibodies isto fuse negatively charged residues to the N- or C-termini. Thus forexample, peptides consisting principally of aspartic acids and glutamicacid may be fused to the N-terminus or C-terminus to the antibody heavychain, light chain or both. Because the N-termini are structurally closeto the antigen binding site, the C-termini are preferred.

Based on the described engineering approaches, a number of variants weredesigned to reduce the isoelectric point of both the antibody heavychain and light chain. The heavy chain variants comprise variouscombinations of isotypic substitutions, as well as C-terminal negativelycharged peptides. Relative to a native IgG1, the variants comprise oneor more isotypic substitutions from the group consisting of G137E,G138S, S192N, L193F, I199T, N203D, K214T, K222T, substitution of 221-225DKTHT to VE, H268Q, K274Q, R355Q, N384S, K392N, V397M, Q419E, and adeletion of K447 (referred to as K447#), wherein numbering is accordingto the EU index. The light chain variants comprise various combinationsof non-isotypic substitutions and C-terminal negatively chargedpeptides. Cκ variants comprise one or more substitutions from the groupconsisting of K126E, K145E, N152D, S156E, K169E, and S202E, whereinnumbering is according to the EU index.

Sequences of the variant heavy chains are provided in FIG. 19, andsequences of the variant light chains are provided in FIG. 20. Table 3lists the variants constructed, along with the calculated pI's of theheavy constant chain, light constant chain, as well as the pI of thefull length monoclonal antibody (mAb) containing the variable region(Fv) of the anti-VEGF antibody Bevacizumab.

TABLE 3 pI-engineered antibody constant chain variants Heavy Chain LightChain Fv mAb^(b) Identity pI Identity pI Identity^(a) VH pI VL pI pIIgG1-WT 8.46 Ck-WT 6.1 Bev 6.99 6.75 8.10 IgG1-WT 8.46 Ck-pI (3) 4.6 Bev6.99 6.75 6.58 IgG1-WT 8.46 Ck-pI (6) 4.4 Bev 6.99 6.75 6.21 IgG1-WT8.46 Ck-pI (6- 4.3 Bev 6.99 6.75 5.85 DEDE) IgG2-WT 7.66 Ck-WT 6.1 Bev6.99 6.75 7.31 IgG2-WT 7.66 Ck-pI (3) 4.6 Bev 6.99 6.75 6.16 IgG2-WT7.66 Ck-pI (6) 4.4 Bev 6.99 6.75 5.88 IgG2-WT 7.66 Ck-pI (6- 4.3 Bev6.99 6.75 5.58 DEDE) pI-iso1 5.93 Ck-WT 6.1 Bev 6.99 6.75 6.16 pI-iso1(NF) 5.93 Ck-WT 6.1 Bev 6.99 6.75 6.16 pI-iso1 (NF-VE) 5.85 Ck-WT 6.1Bev 6.99 6.75 6.11 pI-iso1 (NF-VE) 5.85 Ck-pI (3) 4.6 Bev 6.99 6.75 5.58pI-iso1 (NF-VE) 5.85 Ck-pI (6) 4.4 Bev 6.99 6.75 5.38 pI-iso1 (NF-VE-5.36 Ck-pI (6- 4.3 Bev 6.99 6.75 5.18 DEDE) DEDE) pI-iso1 (NF-VE- 5.36Ck-WT 6.1 Bev 6.99 6.75 5.74 DEDE) pI-iso1 (NF-VE- 5.36 Ck-pI (3) 4.6Bev 6.99 6.75 5.32 DEDE) pI-iso1 (NF-VE- 5.36 Ck-pI (6) 4.4 Bev 6.996.75 5.18 DEDE) pI-iso1 (NF-VE- 5.36 Ck-pI (6- 4.3 Bev 6.99 6.75 5.03DEDE) DEDE) ^(a)Bev = the variable region of the anti-VEGF antibodyBevacizumab ^(b)mAb pI = the pI of the full length monoclonal antibodycontaining the Fv of Bevacizumab

Example 5 Determination of Charge-Dependency of pI Engineering andPotential Combination with Fc Variants that Enhance Binding to FcRn

A series of new pI-engineered variants were generated to test twoaspects of the relationship between low pI and extended half-life.First, the parameter of charge was investigated by making a controlledset of variants based on the 9493 IgG1-pI(12) variant. These variants,10017, 10018, and 10019, are described in Table 4, along with their pIand the differences in positively and negatively charged residuesrelative to bevacizumab IgG1 WT.

TABLE 4 Engineered constructs exploring charge and Fc variants HC HC LCCharge XENP identity Substitutions Substitutions pI State # KR # DE 4547IgG1-WT 8.1 (+6)  0 0 9493 IgG1-pI (12) CH1-pI (6) Ck-pI (6) 5.6 (−30)(−12) (+24) 9992 IgG1-pI (12) CH1-pI (6) + Ck-pI (6) 5.6 (−30) (−12)(+24) N434S 9993 IgG1-pI (12) CH1-pI (6) + Ck-pI (6) 5.6 (−30) (−12)(+24 M428L/N434S 10017 IgG1-pI (6) S119E T164E N152D S156E 6.6 (−6)  0(+12) Neutral-to-DE N208D S202E 10018 IgG1-pI (6)-KR- K133Q K205Q K126QK145Q 6.6 (−6)  (−12) 0 to-Neutral K210Q K169Q 10019 IgG1-pI (6)-KR-K133E K205E K126E K145E 5.9 (−18) (−12) (+12) to-DE K210E K169E CH1-pI(6) = S119E K133E T164E K205E N208D K210E Ck-pI (6) = K126E K145E N152DS156E K169E S202E pI calculated with Fv = Bevacizumab

The experimental rationale here is as follows. If all the mechanism forimproved half-life is based on removal of positive charge, 10018 and10019 should be as good as 9493 while 10017 would not be extended. Ifthe mechanism is based on an increase in negative charge, 10018 will notbe extended, while 10017 and 10019 will have equivalent half-life thatis extended relative to IgG1 but shorter than 9493. If overall pI (orcharge state) is the basis, the result will be 9493>10019>10017=10018.

In addition to the charge-controlled variant set, the 9493 IgG-pI(12)variant was combined with substitutions that improve binding to FcRn atpH 6.0 in order to test whether the two mechanisms of half-lifeimprovement, charge state and FcRn, are compatible. These variants, 9992IgG1-pI(12)-N434S and 9993 IgG1-pI(12)-M428L/N434S, are listed in Table4.

Antibody variants were constructed with the variable region ofbevacizumab using molecular biology techniques as described above.Antibodies were expressed, purified, and characterized as describedabove. PK studies of the variant and control antibodies were carried outin the huFcRn mice as described above. The group mean averages of theserum concentrations are plotted in FIGS. 21 and 22, along with thehalf-lives obtained from the fits of the data.

The results indicate that both reducing positive charge and increasingnegative charge contribute to improved half-life. In addition, theresults indicate that engineered lower pI and increased binding to FcRncan be used in combination to obtain even greater enhancements inhalf-life. A plot of the half-life vs. pI relationship is provided inFIG. 23 for variant and native IgG's of identical Fv (bevacizumab) thathave been tested in the huFcRn mice. The graph illustrates again theinverse relationship between half-life and pI, as well as thecombinability of variants engineered for lower pI and Fc variants thatimprove binding to FcRn.

Example 6 New pI-Engineered Constructs

As described above, efforts can be made to minimize the risk thatsubstitutions that lower pI will elicit immunogenicity by utilizing theisotypic differences between the IgG subclasses (IgG1, IgG2, IgG3, andIgG4). A new set of novel isotypes was designed based on this principal.Again, because immune recognition occurs at a local sequence level, i.e.MHC II and T-cell receptors recognize epitopes typically 9 residues inlength, pI-altering substitutions were accompanied by isotypicsubstitutions proximal in sequence. In this way, epitopes were extendedto match a natural isotype. Such substitutions would thus make upepitopes that are present in other human IgG isotypes, and thus would beexpected to be tolerized.

The designed low-pI isotypes, referred to as IgG-pI-Iso2,IgG-pI-Iso2-SL, IgG-pI-Iso2-charges-only, IgG-pI-Iso3, IgG-pI-Iso3-SL,and IgG-pI-Iso3-charges-only are described in Table 5, along with theirpI and effector function properties. FIG. 24 provides a sequencealignment of IgG-pI-Iso3 with the native IgG isotypes, and depictsresidue identities and residues that reduce pI relative to one or moreof the native IgG isotypes. FIGS. 25 and 26 illustrate the structuraldifferences between IgG1 and IgG-pI-Lso3. IgG-pI-Iso2, IgG-pI-Iso2-SL,and IgG-pI-Iso2-charges-only were designed to have low (weak) effectorfunction, as determined by IgG2-like residues in the hinge (233P, 234V,235A) and CH2 domain (327G). IgG-pI-Iso3, IgG-pI-Iso3-SL, andIgG-pI-Iso3-charges-only were designed to have high (strong) effectorfunction, as determined by IgG1-like residues in the hinge (233E, 234L,235L, 236G) and CH2 domain (327A). Isotypic low pI variants with the“SL” designation indicate that these variants differ from IgG-pI-Iso2and IgG-pI-Iso3 by having 192S and 193L. Serine and leucine at thesepositions were found to be more compatible than 192N/193F due todifferences in neighboring residues that are present in IgG1 and IgG2.Low pI isotype variants designated as “charges only” contain chargeaffecting isotypic substitutions, but do not contain the neighboringnon-charge altering substitutions. The novel isotypes can be combinedwith a native light chain constant region (Ckappa or Clambda), or avariant version engineered with substitutions to further reduce the pI.An example of a pI-engineered light constant chain is a new variantreferred to as CK-pI(4), described schematically in FIG. 27. Inaddition, the novel isotypes can be engineered with Fc variants thatimprove affinity to FcRn, thereby further enabling extended half-life.Such Fc variants may include, for example 434S or 428L/434S as describedin Table 5, or other Fc variants as described herein. Amino acidsequences of IgG-pI-Iso2, IgG-pI-Iso2-SL, IgG-pI-Iso2-charges-only.IgG-pI-Iso3. IgG-pI-Iso3-SL, IgG-pI-Iso3-charges-only and CK-pI(4) areprovided in FIG. 28.

TABLE 5 Novel IgG isotypes with low pI Effector XENP Heavy Light Fc.variant pI Function 10178 IgG-pI-Iso2 WT 6.3 Low 10470 IgG-pI-lso2-SL WT6.3 Low 10180 IgG-pI-Iso2 WT 434S 6.3 Low 10471 IgG-pI-Iso2-SL WT 434S6.3 Low 10182 IgG-pI-Iso2 CK-pI (4) 5.6 Low 10184 IgG-pI-Iso2 CK-pI (4)434S 5.6 Low 10427 IgG-pI-Iso2-charges-only WT 6.3 Low 10473IgG-pI-Iso2-charges-only WT 434S 6.3 Low 10179 IgG-pI-Iso3 WT 6.2 High10286 IgG-pI-Iso3-SL WT 6.2 High 10181 IgG-pI-Iso3 WT 434S 6.2 High10466 IgG-pI-Iso3-SL WT 434S 6.2 High 10467 IgG-pI-Iso3-SL WT 428L/434S6.2 High 10183 IgG-pI-Iso3 CK-pI (4) 5.5 High 10185 IgG-pI-Iso3 CK-pI(4) 434S 5.5 High 10525 IgG-pI-Iso3-SL CK-pI (4) 434S 5.5 High 10426IgG-pI-Iso3-charges-only WT 6.2 High 10472 IgG-pI-Iso3-charges-only WT434S 6.2 High SL = 192S/193L CK-pI (4) = K126E/K145E/K169E/K207E pIcalculated with Fv = Bevacizumab

The novel engineered isotypes can be combined with other Fc variants togenerate antibodies or Fc fusions with extended half-life and otherimproved properties. For example, IgG-pI-Iso2-SL and/or IgG-pI-Iso3-SLmay incorporate variants 239D, 332E, 267E, and/or 328F that modulatebinding to FcγRs to provide enhanced effector function orimmunomodulatory properties. The novel isotypes may be combined withother Fc variants that improve binding to FcRn, including for example428L, 428L; 434S, T250Q/M428L, M252Y/S254T/T256E, and N434A/T307Q,thereby potentially further extending in vivo half-life. Exemplary heavychains are described in Table 6. Such variants may be expressed with alight chain that has a native constant light chain (CK or Cλ), or onethat also incorporates constant light chain modifications that reducepI, including for example any of the engineered constant light chainsdescribed herein, including for example CK-pI(4).

TABLE 6 Engineered combinations of pI isotype variants with othervariants. Heavy Fe IgG-pI-Iso3-SL 332E IgG-pI-Iso3-SL 239D/332EIgG-pI-Iso3-SL 332E/434S IgG-pI-Iso3-SL 239D/332E/434S IgG-pI-Iso2-SL267E/328F IgG-pI-Iso2-SL 434S/267E/328F IgG-pI-Iso3-SL 267E/328FIgG-pI-Iso3-SL 434S/267E/328F IgG-pI-Iso2-SL 428L/434S IgG-pI-Iso3-SL428L/434S IgG-pI-Iso2-SL 428L IgG-pI-Iso3-SL 428L IgG-pI-Iso2-SL250Q/428L IgG-pI-Iso3-SL 250Q/428L IgG-pI-Iso2-SL 252Y/254T/256EIgG-pI-Iso3-SL 252Y/254T/256E IgG-pI-Iso2-SL 434A/307Q IgG-pI-Iso3-SL434A/307Q

In order to reduce pI even further, additional variant heavy constantchains with reduced pI were designed to minimize mutational load byintroducing charge swapping mutations, i.e. where K and R were replacedwith D or E, as described above. To aid in the design of these variants,fraction exposed as well as the energy change upon substitution to Gluwere calculated for each K and R residue in the Fc region (FIG. 29).These new variants are referred to as pI(7) and pI(11). pI(7)incorporated amino acid modifications K133E, K205E, K210E, K274E, R355E,K392E, and a deletion of the Lys at 447, and pI(11) incorporated aminoacid modifications K133E, K205E, K210E, K274E, K320E, K322E, K326E,K334E, R355E, K392E, and a deletion of the Lys at 447 Thesemodifications were introduced into heavy constant chains to result inantibodies with strong effector function, IgG1-pI(7) and IgG1-pI(11),and weak effector function IgG1/2-pI(7) and IgG1/2-pI(11). As can beseen in FIG. 30, as mAb pI gets lower, it requires a greater number ofcharge swap substitutions to decrease pI further. These pI-engineeredvariants are described in Table 7, and amino acid sequences are providedin FIG. 28.

TABLE 7 Engineered charge swaps Fc XENP Heavy variant Light pI 10107IgG1-pI (7) CK-pI (4) 5.3 10108 IgG1-pI (11) CK-pI (4) 5.0 10109IgG1/2-pI (7) CK-pI (4) 5.4 10110 IgG1/2-pI (11) CK-pI (4) 5.0 10476IgG1/2-pI (7) 434S CK-pI (4) 5.4 IgG1-pI (7) =K133E/K205E/K210E/K274E/R355E/K392E/K447# IgG1-pI (11) =K133E/K205E/K210E/K274E/K320E/K322E/K326E/K334E/R355E/K392E/K447#IgG1/2-pI (7) = K133E/K205E/K210E/Q274E/R355E/K392E/K447# IgG1/2-pI (11)= K133E/K205E/K210E/Q274E/K320E/K322E/K326E/K334E/R355E/K392E/K447#CK-pI (4) = K126E/K145E/K169E/K207E pI calculated with Fv = Bevacizumab

Antibody variants were constructed with the variable region ofbevacizumab using molecular biology techniques as described above.Antibodies were expressed, purified, and characterized as describedabove. PK studies of the variant and control antibodies were carried outin the huFcRn mice as described above. The group mean averages of theserum concentrations are plotted in FIG. 31 and FIG. 32, along with thehalf-lives obtained from the fits of the data. Half-lives for individualmice are plotted in FIG. 33. The data clearly demonstrate the additivityof low pI from isotypic pI variants as well as enhanced FcRn bindingfrom the N434S substitution as shown by a plot of half-life vs. pI asshown in FIG. 34.

Example 7 Isotypic Light Chain Constant Region Variants

Homology between CK and Cλ is not as high as between the IgG subclasses(as shown in FIG. 18), however the sequence and structural homology thatexists may still be used to guide substitutions to create an isotypiclow-pI light chain constant region. In FIG. 18, positions with residuescontributing to a higher pI (K, R and H) or lower pI (D and E) arehighlighted in bold. Gray indicates lysine, arginines, and histidinesthat may be substituted, preferably with aspartic or glutatmic acids, tolower the isoelectric point. A structural alignment of CK and Cλ wasconstructed (FIG. 35) and used along with the sequence alignment as aguide to make several CK/Cλ isotypic variants. These pI-engineeredvariants are described in Table 8, and amino acid sequences are providedin FIG. 28.

TABLE 8 Engineered low-pI variants containing isotypic light chainconstant regions Fc Effector XENP Heavy Light variant pI Function 10324IgG-pI-Iso3 CK-Iso (3) 5.9 High 10325 IgG-pI-Iso3 CK-Iso (4) 5.8 High10326 IgG-pI-Iso3 CK-Iso (5) 5.8 High 10327 IgG-pI-Iso3 CK-Iso (6) 5.7High 10511 IgG-pI-Iso3-SL CK-Iso (3) 5.9 High 10512 IgG-pI-Iso3-SLCK-Iso (4) 5.8 High 10513 IgG-pI-Iso3-SL CK-Iso (5) 5.8 High 10517IgG-pI-Iso3-SL CK-Iso (3) 434S 5.9 High 10518 IgG-pI-Iso3-SL CK-Iso (4)434S 5.8 High 10519 IgG-pI-Iso3-SL CK-Iso (5) 434S 5.8 High 10520IgG-pI-Iso3-SL CK-Iso (3) 428L/434S 5.9 High 10521 IgG-pI-Iso3-SL CK-Iso(4) 428L/434S 5.8 High 10522 IgG-pI-Iso3-SL CK-Iso (5) 428L/434S 5.8High 10526 IgG-pI-Iso3 CK-Iso (5) 434S 5.8 High 10527 IgG-pI-Iso2-SLCK-Iso (5) 434S 5.8 Low

Antibody variants were constructed with the variable region ofbevacizumab using molecular biology techniques as described above.Antibodies were expressed, purified, and characterized as describedabove. PK studies of the variant and control antibodies were carried outin the huFcRn mice as described above. The group mean averages of theserum concentrations as well as the half-lives obtained from fits of thedata for one of these variants (XENP10519-IgG-pI-Iso3-SL-434S-CK-Iso(5))are plotted in FIG. 32 and the half-lives for individual mice in FIG.33. This variant is also included in the correlation plot shown in FIG.34. The benefit of lower pI due to the CK-Iso(5) light chain is clearlyshown.

STATEMENT TO SUPPORT SEQUENCE LISTING

This submission is accompanied by a computer readable form containingSEQ ID NOs: 1-212. The sequence listing in computer readable form wasprepared through the use of the software program “PatentIn, version3.5”in accordance with 37 C.F.R. §§1.821-1.825.

According to the Legal Framework for EFS-Web (Sep. 2008), if a sequencelisting text file submitted via EFS-Web complies with the requirementsof 37 C.F.R. §1.824(a)(2)-(6) and (b), the text file will serve as boththe paper copy required by 37 C.F.R. §1.821(c) and the CRF required by37 C.F.R. §1.821(e). Therefore a paper copy of the referenced sequencelisting is not included with this application.

The invention claimed is:
 1. An antibody comprising: a) a variant heavychain constant domain comprising a variant of SEQ ID NO: 2, wherein saidvariant comprises amino acid substitutionsG137E/G138S/I199T/N203D/K214T/K222T/K274Q/Y296F/Y300F/L309V/A339T/R355Q/N384S/K392N/V397M/Q419E/Del447K,and b) a light chain constant domain selected from the group consistingof SEQ ID NOs: 1, 30, 31 and 32, wherein said numbering is according toEU index.
 2. The antibody of claim 1, wherein said light chain constantdomain comprises SEQ ID NO:
 1. 3. The antibody of claim 1, wherein saidlight chain constant domain comprises SEQ ID NO:
 30. 4. The antibody ofclaim 1, wherein said light chain constant domain comprises SEQ ID NO:31.
 5. The antibody of claim 1, wherein said light chain constant domaincomprises SEQ ID NO:
 32. 6. The antibody of claim 1, wherein saidvariant heavy chain constant domain further comprises amino acidsubstitution N434S.
 7. The antibody of claim 1, wherein said variantheavy chain constant domain further comprises amino acid substitutionsM428L/N434S.